Salting Out Genomic DNA Extraction Method - Revision history

Jake Newitt: /* Uses */

2022-03-28T12:36:51Z

Uses

← Older revision		Revision as of 12:36, 28 March 2022
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	==Uses==		==Uses==
	*[[Whole Genome Sequencing]]		*Whole Genome Sequencing
	*[[Template Generation for PCR]]		*Template Generation for PCR

	==Notes==		==Notes==

Jake Newitt: /* Preparation and reagents needed */

2022-03-28T12:36:26Z

Preparation and reagents needed

← Older revision		Revision as of 12:36, 28 March 2022
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	*5ml or 10ml pipette		*5ml or 10ml pipette

	~~'''~~Instructions~~'''~~		==Instructions==
	#Resuspend mycelium in 5ml SET buffer in a 50ml falcon tube. Add 100µl of lysozyme solution. Incubate at 37 degrees C° for 1-3 hours, lysis step is complete when the reaction has clarified and turned viscous. Note that the reaction may not clarify completely, this stage may require organism specific optimisation.		#Resuspend mycelium in 5ml SET buffer in a 50ml falcon tube. Add 100µl of lysozyme solution. Incubate at 37 degrees C° for 1-3 hours, lysis step is complete when the reaction has clarified and turned viscous. Note that the reaction may not clarify completely, this stage may require organism specific optimisation.
	#Add 140µl proteinase K solution and 600µl 10% SDS, then mix by inversion. Incubate at 55° for 2 hours and invert occasionally		#Add 140µl proteinase K solution and 600µl 10% SDS, then mix by inversion. Incubate at 55° for 2 hours and invert occasionally
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	#The solution should be incubated on ice for 2 minutes, before adding 0.6 vol ice cold isopropanol. Mix by inversion and continue to incubate on ice for around 3 minutes. Spool DNA onto a sealed pasteur pipette		#The solution should be incubated on ice for 2 minutes, before adding 0.6 vol ice cold isopropanol. Mix by inversion and continue to incubate on ice for around 3 minutes. Spool DNA onto a sealed pasteur pipette
	#Rinse DNA in 5ml 70% ethanol and leave to air dry. Using cut tips, resuspend in 500µl of TE or sterilised dH<sub>2</sub>O		#Rinse DNA in 5ml 70% ethanol and leave to air dry. Using cut tips, resuspend in 500µl of TE or sterilised dH<sub>2</sub>O


	==Uses==		==Uses==

J Newitt at 12:49, 18 August 2020

2020-08-18T12:49:04Z

← Older revision		Revision as of 12:49, 18 August 2020
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	'''Instructions'''		'''Instructions'''
	#Resuspend mycelium in 5ml SET buffer in a 50ml falcon tube. Add 100µl of lysozyme solution. Incubate at 37 degrees C°		#Resuspend mycelium in 5ml SET buffer in a 50ml falcon tube. Add 100µl of lysozyme solution. Incubate at 37 degrees C° for 1-3 hours, lysis step is complete when the reaction has clarified and turned viscous. Note that the reaction may not clarify completely, this stage may require organism specific optimisation.
	#Add 140µl proteinase K solution and 600µl 10% SDS, then mix by inversion. Incubate at 55° for 2 hours and invert occasionally		#Add 140µl proteinase K solution and 600µl 10% SDS, then mix by inversion. Incubate at 55° for 2 hours and invert occasionally
	#Add 2ml 5M NaCl and mix by inversion		#Add 2ml 5M NaCl and mix by inversion

J Newitt at 15:11, 14 August 2019

2019-08-14T15:11:53Z

← Older revision		Revision as of 15:11, 14 August 2019
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	==Preparation and reagents needed==		==Preparation and reagents needed==
	#Mycelium from 25ml culture (split a 50ml culture into 2)		*Mycelium from 25ml culture (split a 50ml culture into 2)
	#SET buffer:- 75mM NaCl, 25mM EDTA pH8, 20mM Tris-HCl pH7.5		*SET buffer:- 75mM NaCl, 25mM EDTA pH8, 20mM Tris-HCl pH7.5
	#Lysozyme:- 50mg ml<sup>-1</sup>		*Lysozyme:- 50mg ml<sup>-1</sup>
	#Proteinase K:- 20mg ml<sup>-1</sup>		*Proteinase K:- 20mg ml<sup>-1</sup>
	#SDS:- 10%		*SDS:- 10%
	#NaCl:- 5M		*NaCl:- 5M
	#Isopropanol		*Isopropanol
	#70% Ethanol		*70% Ethanol
	#TE buffer of sterilised dH<sub>2</sub>O		*TE buffer of sterilised dH<sub>2</sub>O
	#Sealed Pateur pipette		*Sealed Pateur pipette
	#Eppendorf tubes		*Eppendorf tubes
	#10ml pipettes		*10ml pipettes
	#Yellow tips/blue tips and P200/P1000 pipette		*Yellow tips/blue tips and P200/P1000 pipette
	#5ml or 10ml pipette		*5ml or 10ml pipette

	'''Instructions'''		'''Instructions'''

J Newitt at 14:21, 14 August 2019

2019-08-14T14:21:31Z

← Older revision		Revision as of 14:21, 14 August 2019
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	=Isolation of high molecular weight (HMW) genomic DNA by salting out=		=Isolation of high molecular weight (HMW) genomic DNA by salting out=

	The salting out method is used to isolate high yields of high molecular weight DNA, usually for applications such as whole genome sequencing. It is particularly useful for long read sequencing such as PacBIO and ~~nanopore~~, as ''Streptomyces'' has long regions of ~~repeatitive~~ DNA sequences.		The salting out method is used to isolate high yields of high molecular weight DNA, usually for applications such as whole genome sequencing. It is particularly useful for long read sequencing such as PacBIO and Nanopore, as ''Streptomyces'' has long regions of repetitive DNA sequences.

	It is recommended that a whole day is allocated for this protocol, particularly if it is the first time conducting it.		It is recommended that a whole day is allocated for this protocol, particularly if it is the first time conducting it.
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	==References==		==References==

			Adapted from Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. and Hopwood, D.A., 2000. Practical ''Streptomyces'' genetics (Vol. 291). Norwich: John Innes Foundation.

	Protocol adapted for ActinoBase by Jake T. Newitt, PhD student at the University of East Anglia.		Protocol adapted for ActinoBase by Jake T. Newitt, PhD student at the University of East Anglia.

J Newitt at 14:18, 14 August 2019

2019-08-14T14:18:31Z

← Older revision		Revision as of 14:18, 14 August 2019
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	==References==		==References==


			Protocol adapted for ActinoBase by Jake T. Newitt, PhD student at the University of East Anglia.

Sam Prudence: /* Notes */

2019-08-13T09:54:21Z

Notes

← Older revision		Revision as of 09:54, 13 August 2019
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	==Notes==		==Notes==
	For growth in liquid culture, [[~~DNB~~]] can be made without the agar.		For growth in liquid culture, [[DNA]] can be made without the agar ([[DNB]]).

	==References==		==References==

Sam Prudence: /* Isolation of high molecular weight (HMW) genomic DNA by salting out */

2019-08-13T09:53:07Z

Isolation of high molecular weight (HMW) genomic DNA by salting out

← Older revision		Revision as of 09:53, 13 August 2019
Line 1:		Line 1:
	=Isolation of high molecular weight (HMW) genomic DNA by salting out=		=Isolation of high molecular weight (HMW) genomic DNA by salting out=

	The salting out method is used to isolate high yields of high molecular weight DNA, usually for applications such as whole genome sequencing. It is particularly useful for long read sequencing such as PacBIO and nanopore, as ''Streptomyces'' has long regions of ~~repeating~~ DNA.		The salting out method is used to isolate high yields of high molecular weight DNA, usually for applications such as whole genome sequencing. It is particularly useful for long read sequencing such as PacBIO and nanopore, as ''Streptomyces'' has long regions of repeatitive DNA sequences.

	It is recommended that a whole day is allocated for this protocol, particularly if it is the first time conducting it.		It is recommended that a whole day is allocated for this protocol, particularly if it is the first time conducting it.

Sam Prudence: Sam Prudence moved page Salting Out DNA Extraction Method to Salting Out Genomic DNA Extraction Method

2019-08-13T09:50:37Z

Sam Prudence moved page Salting Out DNA Extraction Method to Salting Out Genomic DNA Extraction Method

← Older revision	Revision as of 09:50, 13 August 2019
(No difference)

Sam Prudence: Sam Prudence moved page Salting out method to Salting Out DNA Extraction Method

2019-08-13T09:48:52Z

Sam Prudence moved page Salting out method to Salting Out DNA Extraction Method

← Older revision	Revision as of 09:48, 13 August 2019
(No difference)