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2020-04-23T22:23:29Z

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=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!
|}

Solutions for Southern Blotting - Revision history

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