ActinoBase - User contributions [en-gb]

Lambda DNA/HindIII Ladder

2020-04-23T22:37:51Z

LisAlgora: Created page with "=Lambda HindIII Marker Preparation= *Lambda (λ) DNA (500 µg/ml): 100µl *10x Reaction Buffer: 45 µl *HindIII or PstI(10U/µl): 5µl *ddWater: 295µl Preparation: #Incubate..."

=Lambda HindIII Marker Preparation=
*Lambda (λ) DNA (500 µg/ml): 100µl
*10x Reaction Buffer: 45 µl
*HindIII or PstI(10U/µl): 5µl
*ddWater: 295µl

Preparation:
#Incubate 90 minutes 37°C
#Add 5µl HindIII or PstI
#Incubate 90 minutes 37°
#Incubate 10 minutes, 75°C
#Store at –20°C

Add 6x loading dye before loading into gel

=Size and Concentration of Bands=
{| class="wikitable"
! style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;;" | Size of Bands in bp (λ HindIII)
! style="font-weight:bold;" | ng in 5µl
|-
| style="font-family:Arial, Helvetica, sans-serif !important;;" | (27491)
| -
|-
| style="font-family:Arial, Helvetica, sans-serif !important;;" | (23130)
| 240
|-
| style="font-family:Arial, Helvetica, sans-serif !important;;" | 9416
| 97
|-
| style="font-family:Arial, Helvetica, sans-serif !important;;" | 6556
| 68
|-
| style="font-family:Arial, Helvetica, sans-serif !important;;" | (4361)
| 44
|-
| style="font-family:Arial, Helvetica, sans-serif !important;;" | 2322
| 24
|-
| style="font-family:Arial, Helvetica, sans-serif !important;;" | 2027
| 21
|-
| style="font-family:Arial, Helvetica, sans-serif !important;;" | 561
| 6
|-
| style="font-family:Arial, Helvetica, sans-serif !important;;" | 125
| -
|}

Southern Blotting

2020-04-23T22:36:18Z

LisAlgora: /* 2. PCR Labelling */

There are many different protocols for Southern blotting. This approach uses vacuum transfer of DNA to a nylon membrane, DIG-labelling of the probe and detection by chromogenic substrate or chemiluminescence.
The buffers for DNA transfer are as described by Sambrook ''et al.'', transfer is carried out by following the instructions for the vacuum pump. The protocols for labelling and detection are as described by the manufacturer’s instructions (Roche Life science in this case).

=Consumables and Solutions=

[[Consumables for Southern Blotting]]

[[Solutions for Southern Blotting]]

=Instructions=

==Labelling the Probe==

If a blot fails completely, it will almost always be because of a bad probe. For best results: label around '''1 µg DNA'''.
Two options for DIG-labelling are described here:

#Random primed DNA labelling: To label a whole cosmid or a fragment of DNA (e.g. fragment excised from a plasmid or a PCR product).You will need a very concentrated DNA sample for this, as each reaction will only label up to 15 µl DNA, and you want approximately 1 µg DNA.

#PCR labelling: Labels during PCR and therefore generates a lot of labelled DNA from small amount of template. This is a more expensive method but, provided PCR has already been optimised, is faster and more likely to generate the required amount of probe.

===1. Random Primed DNA Labelling===
In this method, DNA is made single stranded and then hexanucleotides act as random primers for second strand synthesis using Klenow enzyme. The DIG DNA labelling mix contains dATP. dCTP, dGTP and DIG-labelled dUTP, so DIG is incorporated into the second strand every 20-25 nucleotides as it is synthesised.
#Make volume of DNA sample to 15 µl with water (10 ng – 3µg).
#Boil at 95°C for 10 min to denature DNA (can use a PCR machine for best results), then chill quickly in an ice water bath for around 10 min.
#Set up the following reaction:
#*15 µl denatured DNA
#*2 µl Hexanucleotide mix 10x
#*2 µl DIG DNA labelling mix
#*1 µl Klenow enzyme
#Mix and incubate at 37°C for 1h-20h (for best results, leave overnight)
#Stop the reaction by adding 2 µl 0.2M EDTA (pH 8.0).
#Store at -20°C until required.

===2. PCR Labelling===
The PCR DIG labelling mix contains dATP, dCTP, dGTP and DIG-labelled dUTP, so as the DNA is amplified in a standard PCR reaction DIG is incorporated into every product.
#Set up PCR reaction as usual but instead of using standard dNTPs use PCR DIG labelling mix. Mix is 10x concentrated so use 5 µl per 50 µl reaction.
#Labelled PCR products can be used directly as probe. However, if there are any non-specific products, the correct size product should be excised from an agarose gel and eluted before use.
#You can combine the products of several reactions to use as your probe.
#Store at -20°C until required.

Note: You will need to prepare a probe for the DNA ladder as well in order to have a size reference on the blot. You can prepare your own [[Lambda DNA/HindIII Ladder]] and label with DIG by Random Primed DNA Labelling.

==Electrophoresis==
Important notes:
#Digest DNA samples as required and separate on a 0.6-0.8% agarose gel for 4-6 h or overnight.
#Try to add the same amount of DNA to each lane.
#*If using a positive control, consider the copy number of your target fragment. For example, if you are using digested plasmid as your positive control and digested genomic DNA in your experimental lanes, you will need to add significantly less plasmid DNA than genomic DNA. As a rule of thumb, dilute your plasmid DNA so that you can just barely visualise it on your gel, if you add too much positive control the probe and antibody will be sequestered by this DNA and will give too bright a signal compared to experimental lanes.
#Similarly, dilute your ladder considerably to avoid overdetection of blotted DNA ladder.
#Consider which enzymes to digest with carefully. It will be difficult to differentiate the sizes of large fragments on your blot, so if possible, aim for fragment sizes of <7 kb.
#Be sure to run your DNA far enough through the gel for best resolution of target fragments.
#You can either add ethidium bromide to the gel or stain after electrophoresis. Either way, take a photo as usual (before the blotting) to refer to later.

==Transfer to Membrane==
#Measure the area of the gel that you want to be transferred to the membrane. You can trim the gel so that it has a border around this area of approximately 2 cm. There must be an overlap otherwise vacuum will not form properly.
#Prepare the following before proceeding:
##Cut three sheets of Whatman paper approx. same size as the gel.
##Cut plastic sheeting so that it covers the entire platform of the transfer tray, and cut out a transfer window from the middle of the plastic, exactly the size determined in Step 1.
##Cut membrane so that it overlaps the transfer window by approximately 1 cm all the way round. NB Handle the membrane as little as possible, only at the edges, and only gloved hands.
##Use a pencil to label the back of the membrane, in the top right corner. NB It is very important to keep track of witch side of the membrane is the front.
#Prepare the transfer apparatus as follows:
##Clean the base sheet and place on tray, smooth side up.
##Soak the three sheets of Whatman paper in 2x SSC and place on the centre of base sheet.
##Carefully place the membrane on top (with writing facing down).
##Place plastic sheet over, with transfer window central to the membrane. There should be an overlap of approx. 1 cm. Clip lid of transfer tray into place.
#To check the seal:
##Clean glass sheet with 2x SSC and lay over transfer window.
##Connect transfer tray to pump and switch it on.
##Vacuum pressure should stabilise at around 50mbar. The dial can be used to adjust the pump strength, but if pressure continues to fall it means the seal is faulty. If it happens, it is probably because the plastic sheeting is not covering the platform sufficiently. Take the assembly apart and try a bigger piece of plastic if necessary.
##Switch off pump and remove plate when satisfied that the seal is sufficient to create a vacuum.
#Carefully slide your gel onto the assembly, ensuring that the transfer window aligns to the right section of the gel. Try to avoid air bubbles, rips and tears, but you can remove any air bubbles by smoothing out with a gloved finger.
#Pipette 50 ml depurination buffer onto gel and switch on pump for around 20 min.
#Switch off pump and remove depurination buffer by tilting assembly and then using pipette to remove buffer from corner of tray.
#Pipette 50 ml denaturation buffer onto gel and switch on pump for 20 min.
#Switch off pump and remove buffer as described in Step 7.
#Pipette 50 ml neutralisation buffer onto gel and switch on pump for 20 min.
#Switch off pump and remove buffer as described in Step 7.
#Pipette 50 ml 20x SSC onto gel and switch on pump for around 1 hour to allow DNA transfer to occur.
#Disassemble blot and carefully wrap membrane in clingfilm. Expose to UV for 2 min to crosslink.
#Remove from clingfilm and wash membrane in 2x SSC for 2 min.

==Hybridisation==
#Wrap membrane in a piece of mesh and insert into Hybaid tube. NB If the mesh overlaps with the top of the tube it will break the seal and allow buffer to leak.
#Add 20 ml preheated (~65°C) SHB solution and prehyb in rotating Hybaid oven at 65°C for 1 hour.
#Defrost probe and denature at 100°C for 15 min, then put on ice for 10 min. If reusing old probe, you can denature probe in a Falcon tube, within a beaker of water over a hot plate.
#Decant off old SHB (prehyb) and replace with 20 ml fresh SHB solution plus denatured probe.
#Hybridise overnight at 65°C.

Note: SHB + probe can be stored at -20°C and reused several times when freshly denatured at ~65°C for 10 min before use.

==Stringency Washes==
#Decant SHB and probe from Hybaid tube. The probe can be frozen and reused (store in a Falcon tube).
#Add ~30 ml Stringency Buffer A to Hybaid tube and rinse.
#Wash membrane in Hybaid tube for 15 min with ~150 ml Stringency Buffer A, in Hybaid oven at 65°C.
#Repeat Buffer A wash.
#Add ~30 ml Stringency Buffer B to Hybaid tube and rinse.
#Wash membrane in Hybaid tube for 15 min with ~150 ml Stringency Buffer B, in Hybaid oven at 65°C.
#Repeat Buffer B wash.

==Detection==
#Remove membrane and mesh from Hybaid tube and rinse in Washing buffer for 20s – 30s (no longer).
#Wash for 30 min in 100 ml freshly prepared 1% blocking solution in a clean tray, with gentle rocking or shaking. (This blocks unspecific binding sites on membrane)
#Spin anti-DIG-AP solution for 10 min (reduces speckling on blot).
#Pour off blocking solution and replace with 30 ml antibody solution. Antibody binds to the DIG-labelled probe. Incubate for 30 min with gentle rocking or shaking.
#Decant, and wash in ~100 ml Washing buffer for 15 min to remove unbound antibody.
#Repeat wash.
#Decant washing buffer and equilibrate for 2 min in 20 ml Detection buffer.
#Incubate membrane in 10 ml freshly prepared Colour-substrate solution: 200µl of NBT/BCIP in 10 ml of Detection buffer, kept away from light!), in an appropriate container in the dark. Do not shake during colour development.
#Colour usually develops within a few minutes and the reaction is usually complete after 16 h. The membrane can be exposed to light for short time periods to monitor colour development.
#Stop the reaction when desired spot or band intensities are achieved, by washing the membrane for 5 min with 50 ml of sterile double distilled water or TE-buffer.

Lambda DNA/HindIII ladder

2020-04-23T22:35:20Z

Solutions for Southern Blotting

2020-04-23T22:23:29Z

LisAlgora: Created page with "=Stock Solutions to Prepare in Advance= {| class="wikitable" ! style="font-family:Arial, Helvetica, sans-serif !important;; background-color:rgb(222, 234, 246); color:rgb(0,..."

=Stock Solutions to Prepare in Advance=

{| class="wikitable"
! style="font-family:Arial, Helvetica, sans-serif !important;; background-color:rgb(222, 234, 246); color:rgb(0, 0, 0);" |
! style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; background-color:rgb(222, 234, 246); color:rgb(0, 0, 0);" | Solution
! style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; background-color:rgb(222, 234, 246); color:rgb(0, 0, 0);" | Constituents
! style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; background-color:rgb(222, 234, 246); color:rgb(0, 0, 0);" | Notes
|-
| rowspan="4" style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | For DNA transfer
| style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Depurination solution
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | 19 ml 13M HCl in 1L water
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Add acid to water!
|-
| style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Denaturation solution
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | 87 g NaCl, 20 g NaOH in 1L water
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" |
|-
| style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Neutralisation solution
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | 77.8 g ammonium acetate in 1L water
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" |
|-
| style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | 20x SSC
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | 175.3 g NaCl, 88.2 g sodium citrate in 1L water
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Adjust pH to 7 with HCl. Autoclave
|-
| style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | For probe labelling
| style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | 0.2 M EDTA
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" |
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Adjust pH to 8 with NaOH
|-
| rowspan="4" style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | For Pre-hybridisation and hybridisation
| style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | 10% SDS
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | 10 g SDS in 100 ml water
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Heat to 68°C to help dissolve. Adjust pH to 7.2 with a few drops of conc HCl
|-
| style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | 10% N-laurylsarcosine
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | 5 g in 50 ml water
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" |
|-
| style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Maleic acid buffer
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | 22.2 g maleic acid, 17.6 g NaCl in 1L water
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Adjust pH to 7.5 with NaOH (requires lots, so start off using solid NaOH)
|-
| style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | 10% Blocking solution
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | 5 g blocking reagent in 50 ml maleic acid buffer
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Autoclave to dissolve. Solution remains opaque. Can be stored at 4°C for a few weeks.
|-
| style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | For detection
| style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Detection buffer
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | 12.1 g Tris-HCl, 5.85 g NaCl in 1L water
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Adjust pH to 9.5
|}

=Solutions to Prepare Just Before Use=

{| class="wikitable"
! style="font-weight:bold; background-color:rgb(222, 234, 246); color:rgb(0, 0, 0);" |
! style="background-color:rgb(222, 234, 246); color:rgb(0, 0, 0);" | Solution
! style="font-weight:bold; background-color:rgb(222, 234, 246); color:rgb(0, 0, 0);" | Constituents
! style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; background-color:rgb(222, 234, 246); color:rgb(0, 0, 0);" | Notes
|-
| style="font-weight:bold; color:rgb(0, 0, 0);" | For transfer
| style="font-weight:bold; color:rgb(0, 0, 0);" | 2x SSC
| style="color:rgb(0, 0, 0);" | 20x SSC diluted 10x in water
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | For 1 L: 900 ml water, 100 ml 20 x SSC (Can be stored long term at RT if necessary)
|-
| style="font-weight:bold; color:rgb(0, 0, 0);" | For Pre-hybridisation and Hybridisation
| style="font-weight:bold; color:rgb(0, 0, 0);" | Standard Hybridisation Buffer (SHB)
| style="color:rgb(0, 0, 0);" | 5x SSC, 0.1% N-laurylcarcosine (w/v), 0.02% SDS (w/v), 1% Blocking solution (v/v)
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | For 40 ml (1 blot), add in this order: 26 ml water, 10 ml 20x SSC, 400 μl 10% N-laurylcarcosine, 80 μl 10% SDS, 4 ml 10% Blocking solution
|-
| rowspan="2" style="font-weight:bold; color:rgb(0, 0, 0);" | Stringency washes
| style="font-weight:bold; color:rgb(0, 0, 0);" | Stringency Buffer A
| style="color:rgb(0, 0, 0);" | 2 x SSC, 0.1% SDS
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | In this order: 890 ml water, 100 ml 20x SSC, 10 ml 10% SDS (Can be stored long term at RT if necessary)
|-
| style="font-weight:bold; color:rgb(0, 0, 0);" | Stringency Buffer B
| style="color:rgb(0, 0, 0);" | 0.2 x SSC, 0.1% SDS
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | In this order: 980 ml water, 10 ml 20x SSC, 10 ml 10% SDS (Can be stored long term at RT if necessary)
|-
| rowspan="4" style="font-weight:bold; color:rgb(0, 0, 0);" | For detection
| style="font-weight:bold; color:rgb(0, 0, 0);" | Washing buffer
| style="color:rgb(0, 0, 0);" | 0.3% (w/v) Tween-20 in Maleic acid buffer
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | For 1L: 997 ml Maleic acid buffer, 3 ml Tween-20
|-
| style="font-weight:bold; color:rgb(0, 0, 0);" | 1% Blocking solution
| style="color:rgb(0, 0, 0);" | 10% Blocking solution diluted to 1x Maleic acid buffer
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | For 100 ml: 90 ml Maleic acid buffer, 10 ml 10% Blocking solution
|-
| style="font-weight:bold; color:rgb(0, 0, 0);" | Antibody solution
| style="color:rgb(0, 0, 0);" | Anti-DIG-AP, diluted 1:5000 in 1% Blocking solution
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | For 30 ml (1 blot): 27 ml maleic acid buffer, 3 ml 10% Blocking solution, 3 μl antibody
|-
| style="font-weight:bold; color:rgb(0, 0, 0);" | Colour-substrate solution
| style="color:rgb(0, 0, 0);" | NBT/BCIP solution diluted 50x in Detection buffer
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | For 1 blot: 200 µl of NBT/BCIP solution in 10 ml Detection buffer. Keep in the dark!
|}

Consumables for Southern Blotting

2020-04-23T21:55:28Z

LisAlgora: Created page with "Roche provides a DIG DNA Labelling and Detection Kit that contains: blocking reagent, Anti-DIG-AP conjugate, Hexanucleotide Mix, DIG DNA Labelling Mix, Klenow Enzyme an..."

Roche provides a DIG DNA Labelling and Detection Kit that contains: blocking reagent, Anti-DIG-AP conjugate, Hexanucleotide Mix, DIG DNA Labelling Mix, Klenow Enzyme and NBT/BCIP solution. Otherwise, the components can be purchased separately.

{| class="wikitable"
! style="font-family:Arial, Helvetica, sans-serif !important;; background-color:rgb(222, 234, 246); color:rgb(0, 0, 0);" |
! style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; background-color:rgb(222, 234, 246); color:rgb(0, 0, 0);" | Item
! style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; background-color:rgb(222, 234, 246); color:rgb(0, 0, 0);" | Recommended Supplier
! style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; background-color:rgb(222, 234, 246); color:rgb(0, 0, 0);" | Usage
! style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; background-color:rgb(222, 234, 246); color:rgb(0, 0, 0);" | Storage
|-
| rowspan="4" style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | General
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Hybond N+ membrane (rolls)
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | GE Healthcare
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | ̴ 20 blots
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | RT
|-
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Whatman paper
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | VWR or Fisher
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | ̴ 100 blots
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | RT
|-
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Blocking reagent
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Roche
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | ̴ 25 blots
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | 4°C
|-
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Anti-DIG-AP conjugate
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Roche
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | ̴ 60 reactions
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | -20°C
|-
| rowspan="2" style="font-weight:bold; font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | To label probe
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Either: Hexanucleotide Mix + DIG DNA Labelling Mix +Klenow Enzyme
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Roche
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | 25 to 50 reactions
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | -20°C
|-
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Or: PCR DIG labelling mix + PCR components
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Roche
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | 100 reactions
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | -20°C
|-
| rowspan="2" style="font-weight:bold; color:rgb(0, 0, 0);" | For detection
| style="color:rgb(0, 0, 0);" | Either: NBT/BCIP tablets
| style="color:rgb(0, 0, 0);" | Roche
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | 10-20 blots
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | 4°C
|-
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Or: NBT/BCIP solution
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | Roche
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" |
| style="font-family:Arial, Helvetica, sans-serif !important;; color:rgb(0, 0, 0);" | -20°C
|}

Also required:
**Ammonium acetate
**EDTA
**HCl concetrated
**Maleic acid
**N-laurylsarcosine
**NaCl
**Sodium Citrate
**SDS
**NaOH
**Tris Base
**Tween-20

Southern Blotting

2020-04-23T21:19:14Z

LisAlgora:

There are many different protocols for Southern blotting. This approach uses vacuum transfer of DNA to a nylon membrane, DIG-labelling of the probe and detection by chromogenic substrate or chemiluminescence.
The buffers for DNA transfer are as described by Sambrook ''et al.'', transfer is carried out by following the instructions for the vacuum pump. The protocols for labelling and detection are as described by the manufacturer’s instructions (Roche Life science in this case).

=Consumables and Solutions=

[[Consumables for Southern Blotting]]

[[Solutions for Southern Blotting]]

=Instructions=

==Labelling the Probe==

If a blot fails completely, it will almost always be because of a bad probe. For best results: label around '''1 µg DNA'''.
Two options for DIG-labelling are described here:

#Random primed DNA labelling: To label a whole cosmid or a fragment of DNA (e.g. fragment excised from a plasmid or a PCR product).You will need a very concentrated DNA sample for this, as each reaction will only label up to 15 µl DNA, and you want approximately 1 µg DNA.

#PCR labelling: Labels during PCR and therefore generates a lot of labelled DNA from small amount of template. This is a more expensive method but, provided PCR has already been optimised, is faster and more likely to generate the required amount of probe.

===1. Random Primed DNA Labelling===
In this method, DNA is made single stranded and then hexanucleotides act as random primers for second strand synthesis using Klenow enzyme. The DIG DNA labelling mix contains dATP. dCTP, dGTP and DIG-labelled dUTP, so DIG is incorporated into the second strand every 20-25 nucleotides as it is synthesised.
#Make volume of DNA sample to 15 µl with water (10 ng – 3µg).
#Boil at 95°C for 10 min to denature DNA (can use a PCR machine for best results), then chill quickly in an ice water bath for around 10 min.
#Set up the following reaction:
#*15 µl denatured DNA
#*2 µl Hexanucleotide mix 10x
#*2 µl DIG DNA labelling mix
#*1 µl Klenow enzyme
#Mix and incubate at 37°C for 1h-20h (for best results, leave overnight)
#Stop the reaction by adding 2 µl 0.2M EDTA (pH 8.0).
#Store at -20°C until required.

===2. PCR Labelling===
The PCR DIG labelling mix contains dATP, dCTP, dGTP and DIG-labelled dUTP, so as the DNA is amplified in a standard PCR reaction DIG is incorporated into every product.
#Set up PCR reaction as usual but instead of using standard dNTPs use PCR DIG labelling mix. Mix is 10x concentrated so use 5 µl per 50 µl reaction.
#Labelled PCR products can be used directly as probe. However, if there are any non-specific products, the correct size product should be excised from an agarose gel and eluted before use.
#You can combine the products of several reactions to use as your probe.
#Store at -20°C until required.

Note: You will need to prepare a probe for the DNA ladder as well in order to have a size reference on the blot. You can prepare your own [[Lambda DNA/HindIII ladder]] and label with DIG by Random Primed DNA Labelling.

==Electrophoresis==
Important notes:
#Digest DNA samples as required and separate on a 0.6-0.8% agarose gel for 4-6 h or overnight.
#Try to add the same amount of DNA to each lane.
#*If using a positive control, consider the copy number of your target fragment. For example, if you are using digested plasmid as your positive control and digested genomic DNA in your experimental lanes, you will need to add significantly less plasmid DNA than genomic DNA. As a rule of thumb, dilute your plasmid DNA so that you can just barely visualise it on your gel, if you add too much positive control the probe and antibody will be sequestered by this DNA and will give too bright a signal compared to experimental lanes.
#Similarly, dilute your ladder considerably to avoid overdetection of blotted DNA ladder.
#Consider which enzymes to digest with carefully. It will be difficult to differentiate the sizes of large fragments on your blot, so if possible, aim for fragment sizes of <7 kb.
#Be sure to run your DNA far enough through the gel for best resolution of target fragments.
#You can either add ethidium bromide to the gel or stain after electrophoresis. Either way, take a photo as usual (before the blotting) to refer to later.

==Transfer to Membrane==
#Measure the area of the gel that you want to be transferred to the membrane. You can trim the gel so that it has a border around this area of approximately 2 cm. There must be an overlap otherwise vacuum will not form properly.
#Prepare the following before proceeding:
##Cut three sheets of Whatman paper approx. same size as the gel.
##Cut plastic sheeting so that it covers the entire platform of the transfer tray, and cut out a transfer window from the middle of the plastic, exactly the size determined in Step 1.
##Cut membrane so that it overlaps the transfer window by approximately 1 cm all the way round. NB Handle the membrane as little as possible, only at the edges, and only gloved hands.
##Use a pencil to label the back of the membrane, in the top right corner. NB It is very important to keep track of witch side of the membrane is the front.
#Prepare the transfer apparatus as follows:
##Clean the base sheet and place on tray, smooth side up.
##Soak the three sheets of Whatman paper in 2x SSC and place on the centre of base sheet.
##Carefully place the membrane on top (with writing facing down).
##Place plastic sheet over, with transfer window central to the membrane. There should be an overlap of approx. 1 cm. Clip lid of transfer tray into place.
#To check the seal:
##Clean glass sheet with 2x SSC and lay over transfer window.
##Connect transfer tray to pump and switch it on.
##Vacuum pressure should stabilise at around 50mbar. The dial can be used to adjust the pump strength, but if pressure continues to fall it means the seal is faulty. If it happens, it is probably because the plastic sheeting is not covering the platform sufficiently. Take the assembly apart and try a bigger piece of plastic if necessary.
##Switch off pump and remove plate when satisfied that the seal is sufficient to create a vacuum.
#Carefully slide your gel onto the assembly, ensuring that the transfer window aligns to the right section of the gel. Try to avoid air bubbles, rips and tears, but you can remove any air bubbles by smoothing out with a gloved finger.
#Pipette 50 ml depurination buffer onto gel and switch on pump for around 20 min.
#Switch off pump and remove depurination buffer by tilting assembly and then using pipette to remove buffer from corner of tray.
#Pipette 50 ml denaturation buffer onto gel and switch on pump for 20 min.
#Switch off pump and remove buffer as described in Step 7.
#Pipette 50 ml neutralisation buffer onto gel and switch on pump for 20 min.
#Switch off pump and remove buffer as described in Step 7.
#Pipette 50 ml 20x SSC onto gel and switch on pump for around 1 hour to allow DNA transfer to occur.
#Disassemble blot and carefully wrap membrane in clingfilm. Expose to UV for 2 min to crosslink.
#Remove from clingfilm and wash membrane in 2x SSC for 2 min.

==Hybridisation==
#Wrap membrane in a piece of mesh and insert into Hybaid tube. NB If the mesh overlaps with the top of the tube it will break the seal and allow buffer to leak.
#Add 20 ml preheated (~65°C) SHB solution and prehyb in rotating Hybaid oven at 65°C for 1 hour.
#Defrost probe and denature at 100°C for 15 min, then put on ice for 10 min. If reusing old probe, you can denature probe in a Falcon tube, within a beaker of water over a hot plate.
#Decant off old SHB (prehyb) and replace with 20 ml fresh SHB solution plus denatured probe.
#Hybridise overnight at 65°C.

Note: SHB + probe can be stored at -20°C and reused several times when freshly denatured at ~65°C for 10 min before use.

==Stringency Washes==
#Decant SHB and probe from Hybaid tube. The probe can be frozen and reused (store in a Falcon tube).
#Add ~30 ml Stringency Buffer A to Hybaid tube and rinse.
#Wash membrane in Hybaid tube for 15 min with ~150 ml Stringency Buffer A, in Hybaid oven at 65°C.
#Repeat Buffer A wash.
#Add ~30 ml Stringency Buffer B to Hybaid tube and rinse.
#Wash membrane in Hybaid tube for 15 min with ~150 ml Stringency Buffer B, in Hybaid oven at 65°C.
#Repeat Buffer B wash.

==Detection==
#Remove membrane and mesh from Hybaid tube and rinse in Washing buffer for 20s – 30s (no longer).
#Wash for 30 min in 100 ml freshly prepared 1% blocking solution in a clean tray, with gentle rocking or shaking. (This blocks unspecific binding sites on membrane)
#Spin anti-DIG-AP solution for 10 min (reduces speckling on blot).
#Pour off blocking solution and replace with 30 ml antibody solution. Antibody binds to the DIG-labelled probe. Incubate for 30 min with gentle rocking or shaking.
#Decant, and wash in ~100 ml Washing buffer for 15 min to remove unbound antibody.
#Repeat wash.
#Decant washing buffer and equilibrate for 2 min in 20 ml Detection buffer.
#Incubate membrane in 10 ml freshly prepared Colour-substrate solution: 200µl of NBT/BCIP in 10 ml of Detection buffer, kept away from light!), in an appropriate container in the dark. Do not shake during colour development.
#Colour usually develops within a few minutes and the reaction is usually complete after 16 h. The membrane can be exposed to light for short time periods to monitor colour development.
#Stop the reaction when desired spot or band intensities are achieved, by washing the membrane for 5 min with 50 ml of sterile double distilled water or TE-buffer.

Southern Blotting

2019-09-27T14:35:41Z

LisAlgora: Created page with "There are many different protocols for Southern blotting. This approach uses vacuum transfer pf DNA to a nylon membrane, DIG-labelling of the probe and detection by chromogeni..."

There are many different protocols for Southern blotting. This approach uses vacuum transfer pf DNA to a nylon membrane, DIG-labelling of the probe and detection by chromogenic substrate or chemiluminescence.
The buffers for DNA transfer are as described by Sambrook ''et al.'', transfer is carried out by following the instructions for the vacuum pump. The protocols for labelling and detection are as described by the manufacturer’s instructions (Roche Life science in this case).

=Consumables and Solutions=

[[Consumables for Southern Blotting]]

[[Solutions for Southern Blotting]]

=Instructions=

==Labelling the Probe==

If a blot fails completely, it will almost always be because of a bad probe. For best results: label around '''1 µg DNA'''.
Two options for DIG-labelling are described here:

#Random primed DNA labelling: To label a whole cosmid or a fragment of DNA (e.g. fragment excised from a plasmid or a PCR product).You will need a very concentrated DNA sample for this, as each reaction will only label up to 15 µl DNA, and you want approximately 1 µg DNA.

#PCR labelling: Labels during PCR and therefore generates a lot of labelled DNA from small amount of template. This is a more expensive method but, provided PCR has already been optimised, is faster and more likely to generate the required amount of probe.

===Random Primed DNA Labelling===
In this method, DNA is made single stranded and then hexanucleotides act as random primers for second strand synthesis using Klenow enzyme. The DIG DNA labelling mix contains dATP. dCTP, dGTP and DIG-labelled dUTP, so DIG is incorporated into the second strand every 20-25 nucleotides as it is synthesised.
#Make volume of DNA sample to 15 µl with water (10 ng – 3µg).
#Boil at 95°C for 10 min to denature DNA (can use a PCR machine for best results), then chill quickly in an ice water bath for around 10 min.
#Set up the following reaction:
##15 µl denatured DNA
##2 µl Hexanucleotide mix 10x
##2 µl DIG DNA labelling mix
##1 µl Klenow enzyme
#Mix and incubate at 37°C for 1h-20h (for best results, leave overnight)
#Stop the reaction by adding 2 µl 0.2M EDTA (pH 8.0).
#Store at -20°C until required.

===PCR Labelling===
The PCR DIG labelling mix contains dATP, dCTP, dGTP and DIG-labelled dUTP, so as the DNA is amplified in a standard PCR reaction DIG is incorporated into every product.
#Set up PCR reaction as usual but instead of using standard dNTPs use PCR DIG labelling mix. Mix is 10x concentrated so use 5 µl per 50 µl reaction.
#Labelled PCR products can be used directly as probe. However, if there are any non-specific products, the correct size product should be excised from an agarose gel and eluted before use.
#You can combine the products of several reactions to use as your probe.
#Store at -20°C until required.

==Electrophoresis==

==Transfer to Membrane==

==Hybridisation==

==Stringency Washes==

==Detection==

Nucleic Acid Methods

2019-09-27T13:45:49Z

LisAlgora:

[[ChIP Seq]]

[[RNA extraction from Actinobacteria]]

[[Salting Out Genomic DNA Extraction Method]]

[[Southern Blotting]]

Streptomyces clavuligerus

2019-09-04T14:13:18Z

LisAlgora:

=''[[Streptomyces clavuligerus]]''=

''[[Streptomyces clavuligerus]]'' was originally isolated from a South American soil sample for its ability to produce cephalosporins. Today, it is used in industry for the production of cephamycin C and clavulanic acid (1, 2, 3).
''[[S. clavuligerus]]'' differs from most other ''[[Streptomyces]]'' in that the wildtype is unable to take up sufficient amounts of glucose for it to utilise it as a sole carbon source (4). This has been attributed to the low expression levels of the glucose uptake protein, glucose permease, encoded by ''[[glcP]]'' (5). This is despite the ''[[glcP]]'' gene and the glucokinase gene, ''[[(glk)]]'', being present on the genome. The combination of the glucose permease and glucokinase has been shown to function as the main glucose uptake system in the model organism ''[[S. coelicolor]]''(6,7).

==Cultivation==

Unlike most ''[[Streptomyces]]'', ''[[S. clavuligerus]]'' does not sporulate on [[SFM | MS]] medium but can be grown on [[GYM]] medium for sporulation. Aside from [[GYM]], wildtype grows well on [[TSB]] agar and [[DNA]].

For cultivation in liquid, [[TSB]] medium gives the best result for dispersed growth. For growth curves in liquid, it is best to heat-active spores for 10 minutes at 50° prior to pre-germing for 12 - 24 hours (usually 10 ml of medim). Absorbance measurement readings at 600 nm wavelength can be used up until roughly 48 hours into the growth curve, cell dry weight, however, will give a more accurate result.

==Genome==
The genome of ''S. clavuligerus'' type strain ATCC 2764 has been analysed on several occasions, either by whole genome sequencing (8,9) or PFGE (10). It consists of a chromosome of 6.8 Mb that encodes 25 putative secondary metabolite clusters, including the clavulanic acid and cephamycin C clusters (11), and 4 linear plasmids: pSCL1 (11 kb), pSCL2 (150 kb), pSCL3 (455 kb) and pSCL4 (1,800 b). pSCL4 is a giant linear plasmid and it is the largest linear plasmid published to date and carries 17 putative secondary metabolite clusters including antibiotics such as staurosporine, moenomycin and other β-lactams.

{| class="wikitable"
! style="font-weight:bold; background-color:#cbcefb; color:#333333;" | Replicon
! style="font-weight:bold; background-color:#cbcefb; color:#333333;" | Size (kb)
! style="font-weight:bold; background-color:#cbcefb; color:#333333;" | GC %
! style="font-weight:bold; background-color:#cbcefb; color:#333333;" | Coding sequences
! style="font-weight:bold; background-color:#cbcefb; color:#333333;" | 2ary metabolite clusters
! style="font-weight:bold; background-color:#cbcefb; color:#333333;" | Examples of 2ary metabolites
|-
| style="font-weight:bold;" | Chromosome
| 6,760
| 72.7
| 5,900
| 25
| clavulanic acid, cephamycin C, holomycin
|-
| style="font-weight:bold;" | pSCL1
| 11
| 71.9
| 12
| 0
|
|-
| style="font-weight:bold;" | pSCL2
| 150
| 70.1
| 175
| 0
|
|-
| style="font-weight:bold;" | pSCL3
| 455
| 70.8
| 528
| 1
|
|-
| style="font-weight:bold;" | pSCL4
| 1,796
| 71.9
| 1,728
| 17
| staurosporine, moenomycin
|}

==Protocols==

Preferred media:
* [[GYM]] medium
* [[TSB]] agar and liquid medium
* [[DNA]]

Conjugation

==References==

#Higgens, C. E. and Kastner, R. E. (1971) ‘Streptomyces clavuligerus sp. nov., a β-lactam antibiotic producer’, International Journal of Systematic and Evolutionary Microbiology. Microbiology Society, 21(4), pp. 326–331.
#Howarth, T. T., Brown, A. G. and King, T. J. (1976) ‘Clavulanic acid, a novel β-lactam isolated from Streptomyces clavuligerus; X-ray crystal structure analysis’, Journal of the Chemical Society, Chemical Communications. Royal Society of Chemistry, (7), p. 266b–267.
#Jensen, S. E. and Paradkar, A. S. (1999) ‘Biosynthesis and molecular genetics of clavulanic acid’, Antonie Van Leeuwenhoek. Springer, 75(1), pp. 125–133.
#Garcia-Dominguez, M., Martin, J. F. and Liras, P. (1989) ‘Characterization of sugar uptake in wild-type Streptomyces clavuligerus, which is impaired in glucose uptake, and in a glucose-utilizing mutant.’, Journal of bacteriology. Am Soc Microbiol, 171(12), pp. 6808–6814.
#Pérez-Redondo, R., Santamarta, I., Bovenberg, R., Martín, J. F. and Liras, P. (2010) ‘The enigmatic lack of glucose utilization in Streptomyces clavuligerus is due to inefficient expression of the glucose permease gene’, Microbiology. Microbiology Society, 156(5), pp. 1527–1537.
#Van Wezel, G. P., Mahr, K., König, M., Traag, B. A., Pimentel‐Schmitt, E. F., Willimek, A. and Titgemeyer, F. (2005) ‘GlcP constitutes the major glucose uptake system of Streptomyces coelicolor A3 (2)’, Molecular microbiology. Wiley Online Library, 55(2), pp. 624–636.
#Van Wezel, G. P., König, M., Mahr, K., Nothaft, H., Thomae, A. W., Bibb, M. and Titgemeyer, F. (2007) ‘A new piece of an old jigsaw: glucose kinase is activated posttranslationally in a glucose transport-dependent manner in Streptomyces coelicolor A3 (2)’, Journal of molecular microbiology and biotechnology. Karger Publishers, 12(1–2), pp. 67–74.
#Medema, M.H. et al., 2010. The sequence of a 1.8-Mb bacterial linear plasmid reveals a rich evolutionary reservoir of secondary metabolic pathways. Genome Biology and Evolution, 2(1), pp.212–224.
#Song, J.Y. et al., 2010. Draft genome sequence of Streptomyces clavuligerus NRRL 3585, a producer of diverse secondary metabolites. Journal of Bacteriology, 192(23), pp.6317–6318.
#Netolitzky, D.J. et al., 1995. Giant linear plasmids of beta-lactam antibiotic producing Streptomyces. FEMS Microbiology Letters, 131(1), pp.27–34.
#Chen, C.W. et al., 1994. The linear chromosomes of Streptomyces: structure and dynamics. Actinomycetologica, 8, pp.103–112.

Streptomyces clavuligerus

2019-09-04T14:11:18Z

LisAlgora:

=''[[Streptomyces clavuligerus]]''=

''[[Streptomyces clavuligerus]]'' was originally isolated from a South American soil sample for its ability to produce cephalosporins. Today, it is used in industry for the production of cephamycin C and clavulanic acid (1, 2, 3).
''[[S. clavuligerus]]'' differs from most other ''[[Streptomyces]]'' in that the wildtype is unable to take up sufficient amounts of glucose for it to utilise it as a sole carbon source (4). This has been attributed to the low expression levels of the glucose uptake protein, glucose permease, encoded by ''[[glcP]]'' (5). This is despite the ''[[glcP]]'' gene and the glucokinase gene, ''[[(glk)]]'', being present on the genome. The combination of the glucose permease and glucokinase has been shown to function as the main glucose uptake system in the model organism ''[[S. coelicolor]]''(6,7).

==Cultivation==

Unlike most ''[[Streptomyces]]'', ''[[S. clavuligerus]]'' does not sporulate on [[SFM | MS]] medium but can be grown on [[GYM]] medium for sporulation. Aside from [[GYM]], wildtype grows well on [[TSB]] agar and [[DNA]].

For cultivation in liquid, TSB medium gives the best result for dispersed growth. For growth curves in liquid, it is best to heat-active spores for 10 minutes at 50° prior to pre-germing for 12 - 24 hours (usually 10 ml of medim). Absorbance measurement readings at 600 nm wavelength can be used up until roughly 48 hours into the growth curve, cell dry weight, however, will give a more accurate result.

==Genome==
The genome of ''S. clavuligerus'' type strain ATCC 2764 has been analysed on several occasions, either by whole genome sequencing (8,9) or PFGE (10). It consists of a chromosome of 6.8 Mb that encodes 25 putative secondary metabolite clusters, including the clavulanic acid and cephamycin C clusters (11), and 4 linear plasmids: pSCL1 (11 kb), pSCL2 (150 kb), pSCL3 (455 kb) and pSCL4 (1,800 b). pSCL4 is a giant linear plasmid and it is the largest linear plasmid published to date and carries 17 putative secondary metabolite clusters including antibiotics such as staurosporine, moenomycin and other β-lactams.

{| class="wikitable"
! style="font-weight:bold; background-color:#cbcefb; color:#333333;" | Replicon
! style="font-weight:bold; background-color:#cbcefb; color:#333333;" | Size (kb)
! style="font-weight:bold; background-color:#cbcefb; color:#333333;" | GC %
! style="font-weight:bold; background-color:#cbcefb; color:#333333;" | Coding sequences
! style="font-weight:bold; background-color:#cbcefb; color:#333333;" | 2ary metabolite clusters
! style="font-weight:bold; background-color:#cbcefb; color:#333333;" | Examples of 2ary metabolites
|-
| style="font-weight:bold;" | Chromosome
| 6,760
| 72.7
| 5,900
| 25
| clavulanic acid, cephamycin C, holomycin
|-
| style="font-weight:bold;" | pSCL1
| 11
| 71.9
| 12
| 0
|
|-
| style="font-weight:bold;" | pSCL2
| 150
| 70.1
| 175
| 0
|
|-
| style="font-weight:bold;" | pSCL3
| 455
| 70.8
| 528
| 1
|
|-
| style="font-weight:bold;" | pSCL4
| 1,796
| 71.9
| 1,728
| 17
| staurosporine, moenomycin
|}

==Protocols==

Preferred media:
* [[GYM]] medium
* [[TSB]] agar and liquid medium
* [[DNA]]

Conjugation

==References==

#Higgens, C. E. and Kastner, R. E. (1971) ‘Streptomyces clavuligerus sp. nov., a β-lactam antibiotic producer’, International Journal of Systematic and Evolutionary Microbiology. Microbiology Society, 21(4), pp. 326–331.
#Howarth, T. T., Brown, A. G. and King, T. J. (1976) ‘Clavulanic acid, a novel β-lactam isolated from Streptomyces clavuligerus; X-ray crystal structure analysis’, Journal of the Chemical Society, Chemical Communications. Royal Society of Chemistry, (7), p. 266b–267.
#Jensen, S. E. and Paradkar, A. S. (1999) ‘Biosynthesis and molecular genetics of clavulanic acid’, Antonie Van Leeuwenhoek. Springer, 75(1), pp. 125–133.
#Garcia-Dominguez, M., Martin, J. F. and Liras, P. (1989) ‘Characterization of sugar uptake in wild-type Streptomyces clavuligerus, which is impaired in glucose uptake, and in a glucose-utilizing mutant.’, Journal of bacteriology. Am Soc Microbiol, 171(12), pp. 6808–6814.
#Pérez-Redondo, R., Santamarta, I., Bovenberg, R., Martín, J. F. and Liras, P. (2010) ‘The enigmatic lack of glucose utilization in Streptomyces clavuligerus is due to inefficient expression of the glucose permease gene’, Microbiology. Microbiology Society, 156(5), pp. 1527–1537.
#Van Wezel, G. P., Mahr, K., König, M., Traag, B. A., Pimentel‐Schmitt, E. F., Willimek, A. and Titgemeyer, F. (2005) ‘GlcP constitutes the major glucose uptake system of Streptomyces coelicolor A3 (2)’, Molecular microbiology. Wiley Online Library, 55(2), pp. 624–636.
#Van Wezel, G. P., König, M., Mahr, K., Nothaft, H., Thomae, A. W., Bibb, M. and Titgemeyer, F. (2007) ‘A new piece of an old jigsaw: glucose kinase is activated posttranslationally in a glucose transport-dependent manner in Streptomyces coelicolor A3 (2)’, Journal of molecular microbiology and biotechnology. Karger Publishers, 12(1–2), pp. 67–74.
#Medema, M.H. et al., 2010. The sequence of a 1.8-Mb bacterial linear plasmid reveals a rich evolutionary reservoir of secondary metabolic pathways. Genome Biology and Evolution, 2(1), pp.212–224.
#Song, J.Y. et al., 2010. Draft genome sequence of Streptomyces clavuligerus NRRL 3585, a producer of diverse secondary metabolites. Journal of Bacteriology, 192(23), pp.6317–6318.
#Netolitzky, D.J. et al., 1995. Giant linear plasmids of beta-lactam antibiotic producing Streptomyces. FEMS Microbiology Letters, 131(1), pp.27–34.
#Chen, C.W. et al., 1994. The linear chromosomes of Streptomyces: structure and dynamics. Actinomycetologica, 8, pp.103–112.

R2YE

2019-09-04T13:27:30Z

LisAlgora:

=R2YE=

R2YE medium is a variation of R2 containing yeast extract.

==Preparation==
Make up the following solution:
*103 g Sucrose
*0.25 g K2SO4
*10.12 g MgCl2.6H2O
*10 g Glucose
*0.1 g Difco Casaminoacids
*800 ml dH2O

Pour 80 ml of the solution into 250 ml Erlenmeyer flasks each containing 2.2 g Difco Bacto agar.
Sterilise by autoclaving.
At the time of use, remelt the medium and add to each flask the following autoclaved solutions in the order listed:
*5 ml Difco yeast extract (10%)
*1 ml KH2PO4 (0.5%)
*8 ml CaCl2.2H2O (3.68%)
*1.5 ml L-proline (20%)
*10 ml TES buffer (5.73%, adjusted to pH7.2)
*0.2 ml [[Trace element solution]]
*0.5 ml NaOH (1N) (unsterilised is okay)
*0.75 ml Required growth factors for auxotrophs

==Uses==
*Protoplasts regeneration

==Notes==
''[[Streptomyces clavuligerus]]'' does not utilise glucose and it must be substituted by glycerol.

==References==

Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. and Hopwood, D.A. (2000) Practical Streptomyces Genetics: John Innes Foundation, Norwich Research Park, Colney, Norwich NR4 7UH, UK

Thompson CJ, Ward JM, Hopwood DA (1980) DNA cloning in ''Streptomyces'': resistance genes from antibiotic-producing species. ''Nature'', '''286''', 525-527.

P buffer

2019-09-04T13:25:31Z

LisAlgora:

'''P (protoplast) buffer''' (Okanishi ''et al.'', 1974; Hopwood and Wright, 1978)

Make up the following basal solution:
*103 g Sucrose
*0.25 g K2SO4
*2.02 g MgCl2 . 6H2O
*2 ml [[Trace element solution]]
*800 ml dH2O

Dispense in 80 ml aliquots and autoclave. Before use, add to each flask in order:
*1 ml KH2PO4 (0.5%)
*10 ml CaCl2 . 2H2O (3.68%)
*10 ml TES buffer (5.73%, adjusted to pH7.2)

P buffer

2019-09-04T13:23:05Z

LisAlgora:

'''P (protoplast) buffer''' (Okanishi ''et al.'', 1974; Hopwood and Wright, 1978)

Make up the following basal solution:
*103 g Sucrose
*0.25 g K2SO4
*2.02 g MgCl2.6H2O
*2 ml [[Trace element solution]]
*800 ml dH2O

Dispense in 80 ml aliquots and autoclave. Before use, add to each flask in order:
*1 ml KH2PO4 (0.5%)
*10 ml CaCl2 . 2H2O (3.68%)
*10 ml TES buffer (5.73%, adjusted to pH7.2)

R2

2019-09-04T13:21:53Z

LisAlgora:

=R2=

==Preparation==
Make up the following solution:
*103 g Sucrose
*0.25 g K2SO4
*10.12 g MgCl2.6H2O
*10 g Glucose
*0.1 g Difco Casaminoacids
*800 ml dH2O

Pour 80 ml of the solution into 250 ml Erlenmeyer flasks each containing 2.2 g Difco Bacto agar.
Sterilise by autoclaving.
At the time of use, remelt the medium and add to each flask the following autoclaved solutions in the order listed:
*1 ml KH2PO4 (0.5%)
*8 ml CaCl2.2H2O (3.68%)
*1.5 ml L-proline (20%)
*10 ml TES buffer (5.73%, adjusted to pH7.2)
*0.2 ml [[Trace element solution]]
*0.5 ml NaOH (1N) (unsterilised is okay)
*0.75 ml Required growth factors for auxotrophs

==Uses==
*Protoplasts regeneration

==Notes==
''[[Streptomyces clavuligerus]]'' does not utilise glucose and it must be substituted by glycerol.

==References==

Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. and Hopwood, D.A. (2000) Practical Streptomyces Genetics: John Innes Foundation, Norwich Research Park, Colney, Norwich NR4 7UH, UK

Hopwood DA, Wright HM (1978) Bacterial protoplast fusion-. recombination in fused protoplasts of ''Streptomyces coelicolor''. ''Mol.Gen.Genet.'', '''162''', 307-317

Okanishi M, Suzuki K, Umezawa H (1974) Formation and reversion of streptomycete protoplasts: cultural conditions and morphological study. ''J.Gen.Microbiol.'', '''80''' 389-400

L buffer

2019-09-04T13:21:20Z

LisAlgora:

'''L (lysis) buffer''' (Thompson ''et al.'' 1982)

Mix the following sterile solutions:

*100 ml Sucrose (10.3%)
*10 ml TES buffer (5.73%, adjusted to pH7.2)
*1 ml K2SO4 (2.5%)
*0.2 ml [[Trace element solution]]
*1 ml KH2PO4 (0.5%)
*0.1 ml MgCl2 . 6H2O (2.5M)
*1 ml CaCl2 (0.25M)

This stock solution keeps indefinitely. Just before use dissolve lysozyme in a sample of the solution at a concentration of 1 mg/ml and sterilise by filtration.

Trace element solution

2019-09-04T13:20:59Z

LisAlgora: Created page with "'''Trace element solution (per liter):''' *40 mg ZnCl2 *200 mg FeCl3 . 6H2O *10 mg CuCl2 . 2H2O *10 mg MnCl2..."

'''Trace element solution (per liter):'''
*40 mg ZnCl2
*200 mg FeCl3 . 6H2O
*10 mg CuCl2 . 2H2O
*10 mg MnCl2 . 4H2O
*10 mg Na2B4O7 . 10H2O
*10 mg (NH4)6Mo7O24 . 4H2O

R2

2019-09-04T13:18:58Z

LisAlgora:

L buffer

2019-09-04T13:17:50Z

LisAlgora: Created page with "'''L (lysis) buffer''' (Thompson ''et al.'' 1982) Mix the following sterile solutions: *100 ml Sucrose (10.3%) *10 ml TES buffer (5.73%, adjusted to pH7.2) *1 ml K2</su..."

'''L (lysis) buffer''' (Thompson ''et al.'' 1982)

Mix the following sterile solutions:

*100 ml Sucrose (10.3%)
*10 ml TES buffer (5.73%, adjusted to pH7.2)
*1 ml K2SO4 (2.5%)
*0.2 ml [[Trace element solution]]
*1 ml KH2PO4 (0.5%)
*0.1 ml MgCl2.6H2O (2.5M)
*1 ml CaCl2 (0.25M)

This stock solution keeps indefinitely. Just before use dissolve lysozyme in a sample of the solution at a concentration of 1 mg/ml and sterilise by filtration.

P buffer

2019-09-04T12:51:37Z

LisAlgora: Created page with "'''P (protoplast) buffer''' (Okanishi ''et al.'', 1974; Hopwood and Wright, 1978) Make up the following basal solution: *103 g Sucrose *0.25 g K2SO4 *2...."

'''P (protoplast) buffer''' (Okanishi ''et al.'', 1974; Hopwood and Wright, 1978)

Make up the following basal solution:
*103 g Sucrose
*0.25 g K2SO4
*2.02 g MgCl2.6H2O
*2 ml Trace element solution (Same as [[R2]] and [[R2YE]])
*800 ml dH2O

Dispense in 80 ml aliquots and autoclave. Before use, add to each flask in order:
*1 ml KH2PO4 (0.5%)
*10 ml CaCl2.2H2O (3.68%)
*10 ml TES buffer (5.73%, adjusted to pH7.2)

Cloning, Transformation and Expression of Gene Clusters

2019-09-04T12:40:38Z

LisAlgora:

[[Conjugation using ET12567/pUZ8002]]

[[Heterologous expression of gene clusters]]

[[PAC cloning]]

[[Protoplasts Formation, Regeneration and Transformation]]

[[TAR cloning]]

[[Tri-parental mating]] to generate ET12567 strains for the conjugation of large PAC clones or kanamycin marked vectors into ''Streptomyces''.

Protocols

2019-09-04T12:40:17Z

LisAlgora:

[[File:Actinobase-icons-trans_Protocols.png|150px|Protocols|link=Protocols]]

[[Characterisation of Actinomycetes]]

[[Cloning, Transformation and Expression of Gene Clusters]]

[[CRISPR/Cas9-mediated genome editing]]

[[General Growth and Maintenance of Streptomyces sp.]]

[[High GC PCR]]

[[Isolating Actinomycetes from the environment]]

[[Metabolite extractions]]

[[Microscopy]]

[[Mutagenesis]]

[[Nucleic Acid Methods]]

[[Protein Methods]]

Conjugation using ET12567/pUZ8002

2019-09-04T11:14:00Z

LisAlgora:

Intergeneric transfer of plasmids from ''E. coli'' to ''Streptomyces'' strains was demonstrated after modifying the mobilisation system established by Simon ''et al.'' (1983) for Gram-negative bacteria. It works well also for non-replicative vectors and integrate into the chromosome either site-specifically at the φC31 or pSAM2 attachment sites, or via insert-directed homologous recombination.
Vectors containing ''oriT'' require transfer functions to be supplied by the ''E. coli'' donor strain such as the methylation-deficient ET12567 (MacNeil ''et al.'', 1991) carrying the non-transmissible pUZ8002.

=Materials=
*[[LB]]
*[[2X YT]]
*[[SFM | MS]] plates containing 10 mM MgCl2
*Antibiotics: nalidixic acid (25 mg/ml), chloramphenicol (25 mg/ml), kanamycin (25 mg/ml) and selection antibiotic. See [[Antibiotic stocks and working concentrations]]

===Strains===
*''E. coli'' donor strain ET12567/pUZ8002 containing vector
*Acceptor ''Streptomyces'' strain

=Intructions=

#Transform ET12467/pUZ8002 with ''oriT''-containing vector, and select for the incoming plasmid only
#Inoculate a colony in to 10 ml [[LB]] containing chloramphenicol, kanamycin and the antibiotic used to select for the oriT containing plasmid and grow overnight. Chloramphenicol and kanamycin sensitive segregants arise frequently among transformants, and therefore it is recommended that more than one culture is set up.
#Dilute the overnight culture 1:100 in fresh [[LB]] + antibiotic selection and grow to an OD600 of 0.4-0.6.
#Wash the cells twice with an equal volume of [[LB]] (to remove antibiotics) and resuspend in 0.1 volume of [[LB]]
#While washing the ''E. coli'' cells, for each conjugation add approximately 108 ''Streptomyces'' spores to 500 µl [[2X YT]] broth, heat shock at 50° for 10 minutes then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.
#Add 500 µl of ''E. coli'' cells 500 µl heat-shocked spores or mycelial fragments. Mix and spin briefly. Pour off most of the supernatant and thn resuspend the pellet in the residual liquid.
#Plate out on [[SFM | MS]] agar + 10 mM MgCl2 and incubate at 30° for 16-20 h. Drying the MS for 1 hour in a laminar flow hoof before plating can assist absorption of the solution used in step 9.
#Overlay the plate with 1 ml water containing 0.5 mg nalidixic acid and the plasmid selection antibiotic (e.g 1 mg Apramycin) using a spreader to distribute the antibiotic solution evenly. Continue incubation at 30°
#Pick of potential exconjugants to media containing nalidixic acid and antibiotic selection.

Protoplasts Formation, Regeneration and Transformation

2019-09-04T11:13:36Z

LisAlgora:

Protoplasts are used for fusion, transformation and transfection.

=Protoplasts Preparation=
Although this protocol was written for ''[[Streptomyces lividans]]'' and ''[[Streptomyces coelicolor]]'', many ''Streptomyces'' species can be protoplasted and regenerated using this procedure written by Okanishi ''et al.'' (1974) and adapted by Bibb ''et al.'' (1978) and Thompson ''et al.'' (1982).

Sometimes the age and/or physiological state of the mycelium is important. For example, the best transformation or transfection frequencies in ''S. lividans'' are obtained with protoplasts prepared from mycelium grown to "late exponential" phase (36-40h), whereas for transfection of ''S. parvulus'' protoplasts needed to come from much younger mycelium (c. 18h) The details may also depend on the particular genotype: many morphological mutants are particularly idiosyncratic.

==Materials==
*''Streptomyces'' spores, fresh suspension (c. 109 spores/ml) or frozen stock in 20% glycerol
*[[YEME]] medium
*[[P buffer]]
*10.3% sucrose
*Lysozyme solution (1mg/ml in [[P buffer]], filter sterilised). For most purposes, lysozyme is dissolved in [[P buffer]]; use of [[L buffer]] gives more efficient transformation of ''S. lividans'' by plasmid DNA.
*Autoclaved filter tubes/syringes plugged with cotton wool

==Instructions==

#Add 25 ml [[YEME]] medium to a baffled flask, Add around 0.1 ml spore suspension and required growth factors. Incubate 36-40h at 30° in an orbital incubator shaker. (Cultures of ''S. lividans'' and ''S. coelicolor'' are ready for harvesting when they start to produce red pigment)
#Pour culture broth into a screw cap tube and spin in centrifuge (e.g. 1000x g, 10 min). Discard supernatant
#Re-suspend pellet in 15 ml 10.3% sucrose solution and spin down as above. Discard supernatant
#Repeat step 3. The mycelial pellet, without added liquid, can be frozen at -20° before resuspension.
#Resuspend mycelium in 4 ml lysozyme solution; incubate at 30°, 15-60 min. It is recommended to monitor protoplasts formation using the phase-contrast microscope, as lysozyme treatment times vary from strain to strain, e.g. 15 min is usually long enough for ''S. lividans'' and ''S. clavuligerus'', whereas ''S. coelicolor'' requires 60 min.
#Draw in and out of a 5 ml pipette three times and incubate for a further 15 min.
#Add 5 ml [[P buffer]]. Repeat step 6. This helps to free protoplasts from the mycelium so that they will pass through the cotton wool.
#Filter protoplasts through cotton wool and transfer to a fresh tube.
#Sediment protoplasts gently by spinning in a centrifuge (e.g. 1000x g, 7 min)
#Discard supernatant and suspend protoplasts in 1 ml [[P buffer]].

==Notes==
*Make sure all material (flasks, springs, etc.) is free from detergent. It is recommended to rinse the flasks 3 times in tap water followed by 3 times in distilled water prior to autoclaving
*At any steps when pelleted protoplasts are to be re-suspended, re-suspend in the remaining drop of liquid by tapping the side of the tube repeatedly with a finger until protoplasts are dispersed to form a creamy suspension, then add the suspending [[P buffer]] (otherwise the protoplast pellet is difficult to disperse)
*Avoid vortexing, which induces foaming and consequent lysis
*To freeze the protoplasts for storage, place samples in small plastic tubes and place them in ice in a plastic beaker. Place the beaker at -80° overnight. Free the frozen protoplasts in their tubes from the ice and store at -80°.
*To thaw, shake the frozen tube under running warm water (i.e. ''''freeze slowly, thaw quickly'''')
*To assess the proportion of non-protoplasted units in the suspension, samples can be diluted in parallel in [[P buffer]] and in dilute detergent (e.g. 0.01% SDS) and plated on regeneration plates. Any colonies arising after dilution in detergent are likely to have arisen from non-protoplasted units.

=Further Manipulations of Protoplasts=

[[Protoplast Fusion]]

[[Protoplast Transformation]]

[[Protoplast Transfection]]

=Alternative Methods=

=References=
Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. and Hopwood, D.A. (2000) Practical Streptomyces Genetics: John Innes Foundation, Norwich Research Park, Colney, Norwich NR4 7UH, UK

Conjugation using ET12567/pUZ8002

2019-09-04T10:49:07Z

LisAlgora:

=Strains=
*ET12567/pUZ8002 containing vector
*Donor "Streptomyces" strain

=Intructions=

#Transform ET12467/pUZ8002 with oriT-containing vector, and select for the incoming plasmid only
#Inoculate a colony in to 10 ml LB containing chloramphenicol, kanamycin and the antibiotic used to select for the oriT containing plasmid and grow overnight. Chloramphenicol and kanamycin sensitive segregants arise frequently among transformants, and therefore it is recommended that more than one culture is set up.
#Dilute the overnight culture 1:100 in fresh LB + antibiotic selection and grow to an OD600 of 0.4-0.6.
#Wash the cells twice with an equal volume of LB (to remove antibiotics) and resuspend in 0.1 volume of LB
#While washing the E. coli cells, for each conjugation add approximately 108 Streptomyces spires to 500 µl 2 x YT broth, heat shock at 50° for 10 minutes then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.
#Add 500 µl of E. coli cells 500 µl heat-shocked spores or mycelial fragments. Mix and spin briefly. Pour off most of the supernatant and thn resuspend the pellet in the residual liquid.
#Plate out on MS agar + 10 mM MgCl2 and incubate at 30° for 16-20 h. Drying the MS for 1 hour in a laminar flow hoof before plating can assist absorption of the solution used in step 9.
#Overlay the plate with 1 ml water containing 0.5 mg nalidixic acid and the plasmid selection antibiotic (e.g 1 mg Apramycin) using a spreader to distribute the antibiotic solution evenly. Continue incubation at 30°
#Pick of potential exconjugants to media containing nalidixic acid and antibiotic selection.

Conjugation using ET12567/pUZ8002

2019-09-04T10:46:50Z

LisAlgora:

=Strains=
*ET12567/pUZ8002
*Donor "Streptomyces" strain

=Intructions=

#Transform ET12467/pUZ8002 with oriT-containing vector, and select for the incoming plasmid only
#Inoculate a colony in to 10 ml LB containing Cm, Km and antibiotic used to select for the oriT containing plasmid and grow overnight. Chloramphenicol and Kanamycin sensitive segregants arise frequently among transformants, and therefore it is recommended that more than one culture is set up.
#Dilute the overnight culture 1:100 in fresh LB + antibiotic selection and grow to an OD600 of 0.4-0.6.
#Wash the cells twice with an equal volume of LB (to remove antibiotics) and resuspend in 0.1 volume of LB
#While washing the E. coli cells, for each conjugation add approximately 108 Streptomyces spires to 500 µl 2 x YT broth, heat shock at 50° for 10 minutes then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.
#Add 500 µl of E. coli cells 500 µl heat-shocked spores or mycelial fragments. Mix and spin briefly. Pour off most of the supernatant and thn resuspend the pellet in the residual liquid.
#Plate out on MS agar + 10 mM MgCl2 and incubate at 30° for 16-20 h. Drying the MS for 1 hour in a laminar flow hoof before plating can assist absorption of the solution used in step 9.
#Overlay the plate with 1 ml water containing 0.5 mg nalidixic acid and the plasmid selection antibiotic (e.g 1 mg Apramycin) using a spreader to distribute the antibiotic solution evenly. Continue incubation at 30°
#Pick of potential exconjugants to media containing nalidixic acid and antibiotic selection.

Conjugation using ET12567/pUZ8002

2019-09-04T10:45:08Z

LisAlgora:

=Strains=
*ET12567/pUZ8002
*Donor "Streptomyces" strain

=Intructions=

#Transform ET12467/pUZ8002 with oriT-containing vector, and select for the incoming plasmid only
#Inoculate a colony in to 10 ml LB containing Cm, Km and antibiotic used to select for the oriT containing plasmid and grow overnight. Chloramphenicol and Kanamycin sensitive segregants arise frequently among transformants, and therefore it is recommended that more than one culture is set up.
#Dilute the overnight culture 1:100 in fresh LB + antibiotic selection and grow to an OD600 of 0.4-0.6.
#Wash the cells twice with an equal volume of LB (to remove antibiotics) and resuspend in 0.1 volume of LB
#While washing the E. coli cells, for each conjugation add approximately 108 Streptomyces spires to 500 µl 2 x YT broth, heat shock at 50 oC for 10 minutes then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.
#Add 500 µl of E. coli cells 500 µl heat-shocked spores or mycelial fragments. Mix and spin briefly. Pour off most of the supernatant and thn resuspend the pellet in the residual liquid.
#Plate out on MS agar + 10 mM MgCl2 and incubate at 30 oC for 16-20 h. Drying the MS for 1 hour in a laminar flow hoof before plating can assist absorption of the solution used in step 9.
#Overlay the plate with 1 ml water containing 0.5 mg nalidixic acid and the plasmid selection (e.g 1 mg Apramycin) using a spreader to distribute the antibiotic solution evenly. Continue incubation at 30 oC
#Pick of potential exconjugants to media containing nalidixic acid and antibiotic selection.

Staining of Streptomyces spp. Cell Wall and Nucleic Acids

2019-09-04T10:43:38Z

LisAlgora:

{{DISPLAYTITLE: Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids}}

The following staining technique is a useful method for visualising the actively growing regions of the cell wall, along with the nucleic acids within the cell. The method was developed by Schwedock ''et al.''

This method is well established for ''Streptomyces coelicolor, S. lividans, S. lydicus, S. goldiniensis, S. clavuligerus'' and ''S. collinus''. Furthermore, this protocol can also be used for other Actinomycetes such as ''Pseudonocardia''.

=Method=

==Cultivation of Strains==

Strains should be grown on sterile coverslips (Type 1.5 square coverslips) that have been inserted into agar at a 45° angle. It is advised that the strain of interest should be grown on a [[media]] on which it readily sporulates. Coverslips can be sterilised in ethanol or in an autoclave at 121°C.

After the insertion of the coverslip, 5µl of a 1:100 spore stock dilution is pipetted along the length of the agar-coverslip interface and incubated at the temperature at which the strain grows best (usually 30°C). It is advised to harvest coverslips at two different time points - one before sporulation and one after.

==Reagents==

'''Required Reagents'''

*Fixative - Store at 4°C
*PBS - Store at Room Temperature
*2mg/ml lysozyme in GTE buffer - Store at Room Temperature, shake well before use
*2% BSA in PBS - Store at 4°C
*FITC-WGA staining solution (2µg/ml FITC-WGA, 10µg/ml propidium iodide, 2% BSA in PBS) – stored at 4°C
*Propidium iodide staining solution (10µg/ml PI in PBS) – stored at 4°C
*40% Glycerol in distilled water - Stored at 4°C
*Clear Nail Varnish

'''PBS'''

*NaCl 8g
*KCL 0.2g
*Na2HPO4 1.44g
*KH2PO4 0.24g
*ddH2O 800ml
*pH to 7.4 with HCL and make up to 1000ml ddH2

'''GTE'''

*50mM Glucose
*20mM Tris (pH 8.0)
*20mM EDTA
*Made up in sterile distilled water

'''Stains'''

{| class="wikitable"
! Stains
! Stock
! Working Concentration
! Volume of Stock in 10ml PBS
|-
| Fluorescein-WGA
| 1mg/ml
| 2µg/ml
| 20µl
|-
| Propidium Iodide
| 12.5mg/ml
| 10µg/ml
| 8µl
|}

'''Fixative'''

{| class="wikitable"
!
! Stock Concentration
! Working Concentration
! Volume for 4ml
! Volume for 20ml
|-
| Glutaraldehyde
| 25%
| 0.0045%
| 0.7µl
| 3.5µl
|-
| Formaldehyde
| 40%
| 2.8%
| 280µl
| 1.4ml
|}

(Dilute with PBS)

==Staining==

# Add fixative to each coverslip and incubate at room temperature for 15 minutes.
# Wash coverslips twice with PBS and allow to air dry.
# Rehydrate each sample in PBS for five minutes.
# Cover each coverslip with a solution of 2mg/ml lysosome in GTE for one minute.(1)
# Wash with PBS.
# Incubate with 2% BSA in PBS for five minutes at room temperature.

(1)Strain dependent step, see notes.

In order to prevent photobleaching of stains, complete all following steps in the darkroom.

# Cover each sample in staining solution (2µg/ml fluorescein-WGA, 10µg/ml propidium iodide, 2% BSA in PBS) and incubate for one to three hours at room temperature.
# Wash each sample eight times with 10µg/ml propidium iodide in PBS.
# Remove excess solution and add 8µl of slow fade solution or 40% glycerol in ddH2O.
# Mount the coverslip on a clean glass slide and seal with clear nail varnish.

Protoplasts Formation, Regeneration and Transformation

2019-09-04T10:37:38Z

LisAlgora:

Protoplasts are used for fusion, transformation and transfection.

=Protoplasts Preparation=
Although this protocol was written for ''[[Streptomyces lividans]]'' and ''[[Streptomyces coelicolor]]'', many ''Streptomyces'' species can be protoplasted and regenerated using this procedure written by Okanishi ''et al.'' (1974) and adapted by Bibb ''et al.'' (1978) and Thompson ''et al.'' (1982).

Sometimes the age and/or physiological state of the mycelium is important. For example, the best transformation or transfection frequencies in ''S. lividans'' are obtained with protoplasts prepared from mycelium grown to "late exponential" phase (36-40h), whereas for transfection of ''S. parvulus'' protoplasts needed to come from much younger mycelium (c. 18h) The details may also depend on the particular genotype: many morphological mutants are particularly idiosyncratic.

'''Materials:'''
*''Streptomyces'' spores, fresh suspension (c. 109 spores/ml) or frozen stock in 20% glycerol
*[[YEME]] medium
*[[P buffer]]
*10.3% sucrose
*Lysozyme solution (1mg/ml in [[P buffer]], filter sterilised). For most purposes, lysozyme is dissolved in [[P buffer]]; use of [[L buffer]] gives more efficient transformation of ''S. lividans'' by plasmid DNA.
*Autoclaved filter tubes/syringes plugged with cotton wool

'''Instructions:'''

#Add 25 ml [[YEME]] medium to a baffled flask, Add around 0.1 ml spore suspension and required growth factors. Incubate 36-40h at 30° in an orbital incubator shaker. (Cultures of ''S. lividans'' and ''S. coelicolor'' are ready for harvesting when they start to produce red pigment)
#Pour culture broth into a screw cap tube and spin in centrifuge (e.g. 1000x g, 10 min). Discard supernatant
#Re-suspend pellet in 15 ml 10.3% sucrose solution and spin down as above. Discard supernatant
#Repeat step 3. The mycelial pellet, without added liquid, can be frozen at -20° before resuspension.
#Resuspend mycelium in 4 ml lysozyme solution; incubate at 30°, 15-60 min. It is recommended to monitor protoplasts formation using the phase-contrast microscope, as lysozyme treatment times vary from strain to strain, e.g. 15 min is usually long enough for ''S. lividans'' and ''S. clavuligerus'', whereas ''S. coelicolor'' requires 60 min.
#Draw in and out of a 5 ml pipette three times and incubate for a further 15 min.
#Add 5 ml [[P buffer]]. Repeat step 6. This helps to free protoplasts from the mycelium so that they will pass through the cotton wool.
#Filter protoplasts through cotton wool and transfer to a fresh tube.
#Sediment protoplasts gently by spinning in a centrifuge (e.g. 1000x g, 7 min)
#Discard supernatant and suspend protoplasts in 1 ml [[P buffer]].

'''Notes:'''
*Make sure all material (flasks, springs, etc.) is free from detergent. It is recommended to rinse the flasks 3 times in tap water followed by 3 times in distilled water prior to autoclaving
*At any steps when pelleted protoplasts are to be re-suspended, re-suspend in the remaining drop of liquid by tapping the side of the tube repeatedly with a finger until protoplasts are dispersed to form a creamy suspension, then add the suspending [[P buffer]] (otherwise the protoplast pellet is difficult to disperse)
*Avoid vortexing, which induces foaming and consequent lysis
*To freeze the protoplasts for storage, place samples in small plastic tubes and place them in ice in a plastic beaker. Place the beaker at -80° overnight. Free the frozen protoplasts in their tubes from the ice and store at -80°.
*To thaw, shake the frozen tube under running warm water (i.e. ''''freeze slowly, thaw quickly'''')
*To assess the proportion of non-protoplasted units in the suspension, samples can be diluted in parallel in [[P buffer]] and in dilute detergent (e.g. 0.01% SDS) and plated on regeneration plates. Any colonies arising after dilution in detergent are likely to have arisen from non-protoplasted units.

=Further Manipulations of Protoplats=

[[Protoplast Fusion]]

[[Protoplast Transformation]]

[[Protoplast Transfection]]

Protoplasts Formation, Regeneration and Transformation

2019-09-04T10:37:12Z

LisAlgora:

Protoplasts are used for fusion, transformation and transfection.

=Protoplasts Preparation=
Although this protocol was written for ''[[Streptomyces lividans]]'' and ''[[Streptomyces coelicolor]]'', many ''Streptomyces'' species can be protoplasted and regenerated using this procedure written by Okanishi ''et al.'' (1974) and adapted by Bibb ''et al.'' (1978) and Thompson ''et al.'' (1982).

Sometimes the age and/or physiological state of the mycelium is important. For example, the best transformation or transfection frequencies in ''S. lividans'' are obtained with protoplasts prepared from mycelium grown to "late exponential" phase (36-40h), whereas for transfection of ''S. parvulus'' protoplasts needed to come from much younger mycelium (c. 18h) The details may also depend on the particular genotype: many morphological mutants are particularly idiosyncratic.

'''Materials:'''
*''Streptomyces'' spores, fresh suspension (c. 109 spores/ml) or frozen stock in 20% glycerol
*[[YEME]] medium
*[[P buffer]]
*10.3% sucrose
*Lysozyme solution (1mg/ml in [[P buffer]], filter sterilised). For most purposes, lysozyme is dissolved in [[P buffer]]; use of [[L buffer]] gives more efficient transformation of ''S. lividans'' by plasmid DNA.
*Autoclaved filter tubes/syringes plugged with cotton wool

'''Instructions:'''

#Add 25 ml [[YEME]] medium to a baffled flask, Add around 0.1 ml spore suspension and required growth factors. Incubate 36-40h at 30° in an orbital incubator shaker. (Cultures of ''S. lividans'' and ''S. coelicolor'' are ready for harvesting when they start to produce red pigment)
#Pour culture broth into a screw cap tube and spin in centrifuge (e.g. 1000x g, 10 min). Discard supernatant
#Re-suspend pellet in 15 ml 10.3% sucrose solution and spin down as above. Discard supernatant
#Repeat step 3. The mycelial pellet, without added liquid, can be frozen at -20° before resuspension.
#Resuspend mycelium in 4 ml lysozyme solution; incubate at 30°, 15-60 min. It is recommended to monitor protoplasts formation using the phase-contrast microscope, as lysozyme treatment times vary from strain to strain, e.g. 15 min is usually long enough for ''S. lividans'' and ''S. clavuligerus'', whereas ''S. coelicolor'' requires 60 min.
#Draw in and out of a 5 ml pipette three times and incubate for a further 15 min.
#Add 5 ml [[P buffer]]. Repeat step 6. This helps to free protoplasts from the mycelium so that they will pass through the cotton wool.
#Filter protoplasts through cotton wool and transfer to a fresh tube.
#Sediment protoplasts gently by spinning in a centrifuge (e.g. 1000x g, 7 min)
#Discard supernatant and suspend protoplasts in 1 ml [[P buffer]].

'''Notes:'''
*Make sure all material (flasks, springs, etc.) is free from detergent. It is recommended to rinse the flasks 3 times in tap water followed by 3 times in distilled water prior to autoclaving
*At any steps when pelleted protoplasts are to be re-suspended, re-suspend in the remaining drop of liquid by tapping the side of the tube repeatedly with a finger until protoplasts are dispersed to form a creamy suspension, then add the suspending [[P buffer]] (otherwise the protoplast pellet is difficult to disperse)
*Avoid vortexing, which induces foaming and consequent lysis
*To freeze the protoplasts for storage, place samples in small plastic tubes and place them in ice in a plastic beaker. Place the beaker at -80° overnight. Free the frozen protoplasts in their tubes from the ice and store at -80°.
*To thaw, shake the frozen tube under running warm water (i.e. ''''freeze slowly, thaw quickly''')
*To assess the proportion of non-protoplasted units in the suspension, samples can be diluted in parallel in [[P buffer]] and in dilute detergent (e.g. 0.01% SDS) and plated on regeneration plates. Any colonies arising after dilution in detergent are likely to have arisen from non-protoplasted units.

=Further Manipulations of Protoplats=

[[Protoplast Fusion]]

[[Protoplast Transformation]]

[[Protoplast Transfection]]

Protoplasts Formation, Regeneration and Transformation

2019-08-30T15:31:30Z

LisAlgora:

Protoplasts Formation, Regeneration and Transformation

2019-08-30T15:30:51Z

LisAlgora:

Protoplasts Formation, Regeneration and Transformation

2019-08-30T15:28:57Z

LisAlgora: Created page with "Protoplasts are used for fusion, transformation and transfection. =Protoplasts Preparation= Although this protocol was written for ''Streptomyces lividans'' and ''Str..."

Protocols

2019-08-30T14:29:57Z

LisAlgora:

[[File:Actinobase-icons-trans_Protocols.png|150px|Protocols|link=Protocols]]

[[Characterisation of Actinomycetes]]

[[Cloning, Transformation and Expression of Gene Clusters]]

[[CRISPR/Cas9-mediated genome editing]]

[[General Growth and Maintenance of Streptomyces sp.]]

[[High GC PCR]]

[[Isolating Actinomycetes from the environment]]

[[Microscopy]]

[[Mutagenesis]]

[[Nucleic Acid Methods]]

[[Protein Methods]]

[[Protoplasts Formation, Regeneration and Transformation]]

PIJ6902

2019-08-30T14:12:08Z

LisAlgora:

{{DISPLAYTITLE: pIJ6092}}

==Use==
Chromosomally integrating P''tipA'' expression vector

==Features==
*Apramycin and Thiostrepton resistance cassettes
*φC31 integration site & integrase
*Thiostrepton-inducible ''tipA'' promoter

==History==

==Map==
[[File:pIJ6902.gif]]

==Sequence links==

==References==
Huang, J. , Shi, J. , Molle, V. , Sohlberg, B. , Weaver, D. , Bibb, M. J., Karoonuthaisiri, N. , Lih, C. , Kao, C. M., Buttner, M. J. and Cohen, S. N. (2005), Cross‐regulation among disparate antibiotic biosynthetic pathways of Streptomyces coelicolor. Molecular Microbiology, 58: 1276-1287.

PIJ6902

2019-08-30T14:07:57Z

LisAlgora:

{{DISPLAYTITLE: pIJ6092}}

==Use==
Chromosomally integrating P''tipA'' expression vector

==Features==
*Apramycin and Thiostrepton resistance cassettes
*φC31 integration site & integrase
*Thiostrepton-inducible ''tipA'' promoter

==History==

==Map==
[[File:pIJ6902.gif]]

==Sequence links==

==References==
Huang J., Shi J., Molle V., Sohlberg B., Weaver D., Bibb M.J. Cross-regulation among disparate antibiotic biosynthetic pathways of Streptomyces coelicolor. Mol. Microbiol. 2005;58:1276–1287

PIJ6902

2019-08-30T14:00:35Z

LisAlgora:

{{DISPLAYTITLE: pIJ6092}}

==Use==
Chromosomally integrating P''tipA'' expression vector

==Features==
*Apramycin and Thiostrepton resistance cassettes
*φC31 integration site & integrase

==History==

==Map==
[[File:pIJ6902.gif]]

==Sequence links==

==References==
Huang J., Shi J., Molle V., Sohlberg B., Weaver D., Bibb M.J. Cross-regulation among disparate antibiotic biosynthetic pathways of Streptomyces coelicolor. Mol. Microbiol. 2005;58:1276–1287

PIJ6902

2019-08-30T13:59:57Z

LisAlgora:

{{DISPLAYTITLE: pIJ6092}}

==Use==
Chromosomally integrating PtipA expression vector

==Features==
*Apramycin and Thiostrepton resistance cassettes
*φC31 integration site & integrase

==History==

==Map==
[[File:pIJ6902.gif]]

==Sequence links==

==References==
Huang J., Shi J., Molle V., Sohlberg B., Weaver D., Bibb M.J. Cross-regulation among disparate antibiotic biosynthetic pathways of Streptomyces coelicolor. Mol. Microbiol. 2005;58:1276–1287

PIJ6902

2019-08-30T13:56:51Z

LisAlgora:

{{DISPLAYTITLE: pIJ6092}}

==Use==
Chromosomally integrating PtipA expression vector

==Features==
*Apramycin and Thiostrepton resistance cassettes
*φC31 integration site & integrase

==History==

==Map==
[[File:pIJ6902.jpg]]

==Sequence links==

==References==
Huang J., Shi J., Molle V., Sohlberg B., Weaver D., Bibb M.J. Cross-regulation among disparate antibiotic biosynthetic pathways of Streptomyces coelicolor. Mol. Microbiol. 2005;58:1276–1287

File:PIJ6902.gif

2019-08-30T13:53:21Z

LisAlgora:

PIJ6902

2019-08-30T13:48:08Z

LisAlgora: Created page with "{{DISPLAYTITLE: pIJ6092}} ==Use== ==Features== *Apramycin resistance cassette *φBT1 integration site & integrase ==History== ==Map== ==Sequence links== ==References=..."

{{DISPLAYTITLE: pIJ6092}}

==Use==

==Features==
*Apramycin resistance cassette
*φBT1 integration site & integrase

==History==

==Map==

==Sequence links==

==References==
Huang J., Shi J., Molle V., Sohlberg B., Weaver D., Bibb M.J. Cross-regulation among disparate antibiotic biosynthetic pathways of Streptomyces coelicolor. Mol. Microbiol. 2005;58:1276–1287

Plasmids

2019-08-30T13:43:56Z

LisAlgora:

[[File:Actinobase-icons-trans_Plasmids.png|150px|Plasmids|link=Plasmids]]

Various plasmids have been developed for use in Streptomyces (most will also contain an origin of replication that functions in Escherichia coli, for cloning, general maintenance, and [[intergeneric conjugation into Streptomyces]]). Many of the plasmids commonly used for Streptomyces work are '''integrative''': they insert into a specific site on the Streptomyces chromosome using a [[site-specific phage recombinase]].

The following is an alphabetical list of a number of useful vectors - see also [[Overview of Streptomyces vectors]].

==Plasmids used in Streptomyces==

[[pCAP03]]

[[pCRISPomyces-2]]

[[pESAC13]]

[[pIJ101]]

[[pIJ8660]]

[[pIJ10257]]

[[pIJ12738]]

[[pIJ12742]]

[[pIJ6902]]

[[pKC1132]]

[[pMF96]]

[[pMS82]]

[[pRT802]]

[[pSET152]]

[[pSS170]]

[[Supercos1]]

[[pGM9]]

[[pGM190]]

==Plasmids used for PCR targeting (Redirect)==

[http://streptomyces.org.uk/redirect/index.html Redirect protocol and template information]

[[pIJ773]]

==Plasmids used for heterologous protein expression in E. coli==

The following plasmids are frequently used to express proteins heterologously in E. coli, e.g. for two-hybrid analysis or protein purification.

[[pET28a]]

Vectors for Bacterial two-hybrid experiments
[[pKT25/pKNT25]] | [[pUT18/pUT18C]]

SC

2019-07-26T17:33:14Z

LisAlgora:

=Starch Casein Agar=

==Preparation==
Per Litre:
*10 g soluble starch
*2 g K2HPO4
*2 g KNO3
*0.3 g casein
*0.05 g MgSO4*7H2O
*0.02 g CaCO3
*0.01 g FeSO4*7H2O
*15 g agar

'''Instructions'''
#Mix all the components together well, and adjust the volume with distilled water to 1 L final volume.
#Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
#Mix well and pour into sterile Petri dishes.

==Uses==
*[[Isolation experiments]]

==Notes==

==References==

Streptomyces

2019-07-26T17:26:30Z

LisAlgora:

[https://en.wikipedia.org/wiki/Streptomyces Streptomyces] bacteria comprise at least 600 known species and thousands of strain variants. They are filamentous, sporulating bacteria best know for their prolific production of secondary metabolites, many of which are used in human medicine. [https://en.wikipedia.org/wiki/Selman_Waksman Selman Waksman] made them famous by identifying the first antibiotics from these bacteria, including [https://en.wikipedia.org/wiki/Streptomycin streptomycin] which is used to treat [https://www.who.int/news-room/fact-sheets/detail/tuberculosis tuberculosis], but the field of Streptomyces genetics was founded by [https://en.wikipedia.org/wiki/David_Hopwood Sir David Hopwood FRS] when he moved to Norwich to become John Innes Professor of Genetics at the newly established [http://www.uea.ac.uk University of East Anglia] in 1968. He set up his lab at the [http://www.jic.ac.uk John Innes Centre] , which had moved to Norwich to forge links with UEA, and recruited [https://en.wikipedia.org/wiki/Keith_Chater Keith Chater], [https://en.wikipedia.org/wiki/Mervyn_Bibb Mervyn Bibb] and [https://www.jic.ac.uk/people/professor-mark-buttner/ Mark Buttner] who have all made outstanding contributions to the field and trained many of the scientists who now work on Streptomyces bacteria all over the world.

Essential reading on the taxonomy of streptomycetes: [https://www.hutchingslab.uk/Downloads/Kampfer.pdf Kämpfer et al (2014) The Family Streptomycetaceae, in The Prokaryotes – Actinobacteria]

A fascinating account of the history of Streptomyces research by Sir David Hopwood FRS: [https://global.oup.com/academic/product/streptomyces-in-nature-and-medicine-9780195150667?cc=gb&lang=en& Streptomyces in nature and medicine]

[[File:Streptomyces.png | ''Streptomyces coelicolor'' growing on SFM agar | thumb]]

[[Streptomyces albidoflavus]] strains are found all over the world in soil and marine habitats and associated with insects.

[[Streptomyces avermitilis]] - known for producing ivermectin.

[[Streptomyces clavuligerus]] - known for producing clavulanic acid.

[[Streptomyces coelicolor]] - a model organism for more than 50 years and the first to be sequenced in 2002.

[[Streptomyces formicae]] - known for making formicamycins.

[[Streptomyces goldiniensis]] - known for producing aurodox.

[[Streptomyces griseus]] - known for producing streptomycin.

[[Streptomyces venezuelae]] - a model organism that completes its life cycle in 24 hours and sporulates in liquid culture.

ISP2

2019-07-26T17:22:39Z

LisAlgora:

=International ''Streptomyces'' Project-2 Medium (ISP-2)=

ISP-2 is a rich medium that is routinely used for cultivating ''Streptomyces lydicus'' along with various other plant root associated strains. It contains malt extract and yeast extract, with dextrose as a carbon source; exact composition will vary between batches.

Many ''Streptomyces'' spp. grow well on ISP-2, but developmental and/or antibiotic production phenotypes may differ compared to growth on other media (e.g. [[SFM]]).

==Preparation==
Per Litre:
*4 g Yeast extract powder
*10 g Malt extract powder
*4 g Dextrose
*20 g Agar
*1 L dH2O

'''Instructions'''
#For solid medium, add 2g of Agar to each flask
#Add the rest of the components to a beaker with the dH2O
#Add 100ml to each flask
#Autoclave

==Uses==
*[[Streptomyces maintenance]]
*[[Spore titres]]
*[[Re-isolation experiments]]
*[[Selection for resistance to salt-sensitive antibiotics]]

==Notes==
For growth in liquid culture, [[ISP-2]] can be made without the agar.

==References==

R2

2019-07-26T17:16:54Z

LisAlgora:

=R2=

==Preparation==
Make up the following solution:
*103 g Sucrose
*0.25 g K2SO4
*10.12 g MgCl2.6H2O
*10 g Glucose
*0.1 g Difco Casaminoacids
*800 ml dH2O

Pour 80 ml of the solution into 250 ml Erlenmeyer flasks each containing 2.2 g Difco Bacto agar.
Sterilise by autoclaving.
At the time of use, remelt the medium and add to each flask the following autoclaved solutions in the order listed:
*1 ml KH2PO4 (0.5%)
*8 ml CaCl2.2H2O (3.68%)
*1.5 ml L-proline (20%)
*10 ml TES buffer (5.73%, adjusted to pH7.2)
*0.2 ml 1Trace element solution
*0.5 ml NaOH (1N) (unsterilised is okay)
*0.75 ml Required growth factors for auxotrophs

1Trace element solution(l-1):
**40 mg ZnCl2
**200 mg FeCl3.6H2O
**10 mg CuCl2.2H2O
**10 mg MnCl2.4H2O
**10 mg Na2B4O7.10H2O
**10 mg (NH4)6Mo7O24.4H2O

==Uses==
*Protoplasts regeneration

==Notes==
''[[Streptomyces clavuligerus]]'' does not utilise glucose and it must be substituted by glycerol.

==References==

Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. and Hopwood, D.A. (2000) Practical Streptomyces Genetics: John Innes Foundation, Norwich Research Park, Colney, Norwich NR4 7UH, UK

Hopwood DA, Wright HM (1978) Bacterial protoplast fusion-. recombination in fused protoplasts of ''Streptomyces coelicolor''. ''Mol.Gen.Genet.'', '''162''', 307-317

Okanishi M, Suzuki K, Umezawa H (1974) Formation and reversion of streptomycete protoplasts: cultural conditions and morphological study. ''J.Gen.Microbiol.'', '''80''' 389-400

R2YE

2019-07-26T17:16:02Z

LisAlgora: Created page with "=R2YE= R2YE medium is a variation of R2 containing yeast extract. ==Preparation== Make up the following solution: *103 g Sucrose *0.25 g K2SO4 *10.12 g..."

=R2YE=

R2YE medium is a variation of R2 containing yeast extract.

==Preparation==
Make up the following solution:
*103 g Sucrose
*0.25 g K2SO4
*10.12 g MgCl2.6H2O
*10 g Glucose
*0.1 g Difco Casaminoacids
*800 ml dH2O

Pour 80 ml of the solution into 250 ml Erlenmeyer flasks each containing 2.2 g Difco Bacto agar.
Sterilise by autoclaving.
At the time of use, remelt the medium and add to each flask the following autoclaved solutions in the order listed:
*5 ml Difco yeast extract (10%)
*1 ml KH2PO4 (0.5%)
*8 ml CaCl2.2H2O (3.68%)
*1.5 ml L-proline (20%)
*10 ml TES buffer (5.73%, adjusted to pH7.2)
*0.2 ml 1Trace element solution
*0.5 ml NaOH (1N) (unsterilised is okay)
*0.75 ml Required growth factors for auxotrophs

1Trace element solution(l-1):
**40 mg ZnCl2
**200 mg FeCl3.6H2O
**10 mg CuCl2.2H2O
**10 mg MnCl2.4H2O
**10 mg Na2B4O7.10H2O
**10 mg (NH4)6Mo7O24.4H2O

==Uses==
*Protoplasts regeneration

==Notes==
''[[Streptomyces clavuligerus]]'' does not utilise glucose and it must be substituted by glycerol.

==References==

Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. and Hopwood, D.A. (2000) Practical Streptomyces Genetics: John Innes Foundation, Norwich Research Park, Colney, Norwich NR4 7UH, UK

Thompson CJ, Ward JM, Hopwood DA (1980) DNA cloning in ''Streptomyces'': resistance genes from antibiotic-producing species. ''Nature'', '''286''', 525-527.

R2

2019-07-26T17:01:17Z

LisAlgora:

=R2=

==Preparation==
Make up the following solution:
*103 g Sucrose
*0.25 g K2SO4
*10.12 g MgCl2.6H2O
*10 g Glucose
*0.1 g Difco Casaminoacids
*800 ml dH2O

Pour 80 ml of the solution into 250 ml Erlenmeyer flasks each containing 2.2 g Difco Bacto agar.
Sterilise by autoclaving.
At the time of use, remelt the medium and add to each flask the following autoclaved solutions in the order listed:
*1 ml KH2PO4 (0.5%)
*8 ml CaCl2.2H2O (3.68%)
*1.5 ml L-proline (20%)
*10 ml TES buffer (5.73%, adjusted to pH7.2)
*0.2 ml 1Trace element solution
*0.5 ml NaOH (1N) (unsterilised is okay)
*0.75 ml Required growth factors for auxotrophs

1Trace element solution(l-1):
**40 mg ZnCl2
**200 mg FeCl3.6H2O
**10 mg CuCl2.2H2O
**10 mg MnCl2.4H2O
**10 mg Na2B4O7.10H2O
**10 mg (NH4)6Mo7O24.4H2O

==Uses==
*Protoplasts regeneration

==Notes==
''[[Streptomyces clavuligerus]]'' does not utilise glucose and so, it must be substituted by glycerol

==References==

Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. and Hopwood, D.A. (2000) Practical Streptomyces Genetics: John Innes Foundation, Norwich Research Park, Colney, Norwich NR4 7UH, UK

Hopwood DA, Wright HM (1978) Bacterial protoplast fusion-. recombination in fused protoplasts of ''Streptomyces coelicolor''. ''Mol.Gen.Genet.'', '''162''', 307-317

Okanishi M, Suzuki K, Umezawa H (1974) Formation and reversion of streptomycete protoplasts: cultural conditions and morphological study. ''J.Gen.Microbiol.'', '''80''' 389-400

ISP3

2019-07-26T16:59:46Z

LisAlgora:

=International ''Streptomyces'' Project-3 Medium (ISP-3)=
International Streptomyces Project Medium 3 (ISP-3) is a rich general use media. Alternatively called oatmeal agar.

==Preparation==
*20 g White Oats
*18 g Agar
*1 mL Trace salts solution
**0.1 g FeSO4 x 7H2O
**0.1 g MnCl2 x 4H2O
**0.1 g ZnSO4 x 7H2O
**100 mL dH2O
*1 L dH2O

'''Instructions'''
#Boil 20 g oats in 1L dH2O for 20 mins
#Filter oats through cheesecloth and top volume up to 1L
#Adjust pH to 7.2, add agar, and autoclave
#Add 1 mL Trace Salts solution

==Uses==
*Inducing sporulation of environmental isolates
*Screening for antibiotic production

==Notes==
When screening for antibiotic production, there can be significant differences between media prepared in-house and bought in media

==References==

ISP2

2019-07-26T16:58:49Z

LisAlgora:

=International ''Streptomyces'' Project-2 Medium (ISP-2)=

ISP-2 is a rich medium that is routinely used for cultivating Streptmyces lydicus along with various other plant root associated strains. It contains malt extract and yeast extract, with dextrose as a carbon source; exact composition will vary between batches.

Many ''Streptomyces'' spp. grow well on ISP-2, but developmental and/or antibiotic production phenotypes may differ compared to growth on other media (e.g. [[SFM]]).

==Preparation==
Per Litre:
*4 g Yeast extract powder
*10 g Malt extract powder
*4 g Dextrose
*20 g Agar
*1 L dH2O

'''Instructions'''
#For solid medium, add 2g of Agar to each flask
#Add the rest of the components to a beaker with the dH2O
#Add 100ml to each flask
#Autoclave

==Uses==
*[[Streptomyces maintenance]]
*[[Spore titres]]
*[[Re-isolation experiments]]
*[[Selection for resistance to salt-sensitive antibiotics]]

==Notes==
For growth in liquid culture, [[ISP-2]] can be made without the agar.

==References==

ISP2

2019-07-26T16:57:23Z

LisAlgora:

=International Streptomyces Project-2 Medium (ISP-2)=

ISP-2 is a rich medium that is routinely used for cultivating Streptmyces lydicus along with various other plant root associated strains. It contains malt extract and yeast extract, with dextrose as a carbon source; exact composition will vary between batches.

Many ''Streptomyces'' spp. grow well on ISP-2, but developmental and/or antibiotic production phenotypes may differ compared to growth on other media (e.g. [[SFM]]).

==Preparation==
Per Litre:
*4 g Yeast extract powder
*10 g Malt extract powder
*4 g Dextrose
*20 g Agar
*1 L dH2O

'''Instructions'''
#For solid medium, add 2g of Agar to each flask
#Add the rest of the components to a beaker with the dH2O
#Add 100ml to each flask
#Autoclave

==Uses==
*[[Streptomyces maintenance]]
*[[Spore titres]]
*[[Re-isolation experiments]]
*[[Selection for resistance to salt-sensitive antibiotics]]

==Notes==
For growth in liquid culture, [[ISP-2]] can be made without the agar.

==References==