Cesium Chloride Preparation of DNA - Revision history

Davebrid: updated cesium concentration

2012-01-17T22:03:11Z

updated cesium concentration

← Older revision		Revision as of 22:03, 17 January 2012
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	#Load into two ultracentrifuge tubes then add 25 uL EtBR to each tube		#Load into two ultracentrifuge tubes then add 25 uL EtBR to each tube
	#Centrifuge 60 000 RPM (250 000g) O/N in NVT90 rotor		#Centrifuge 60 000 RPM (250 000g) O/N in NVT90 rotor
	#Withdraw the upper band with a 18g needle and reload into 1g/L CsCl for second spin		#Withdraw the upper band with a 18g needle and reload into 0.1g/mL CsCl for second spin
	#Remove band and extract EtBr with butanol until clear		#Remove band and extract EtBr with butanol until clear
	#Dialyse overnight in 2L of TE		#Dialyse overnight in 2L of TE
	#Precipitate with 3 vol absolute ethanol and 1/10 vol of 3M Acetate pH 7		#Precipitate with 3 vol absolute ethanol and 1/10 vol of 3M Acetate pH 7
	#Resuspend to a final concentration of 5-10 mg/mL in TE		#Resuspend to a final concentration of 5-10 mg/mL in TE

Davebrid: added concentration to Acetate Buffer

2010-03-08T16:14:26Z

added concentration to Acetate Buffer

← Older revision		Revision as of 16:14, 8 March 2010
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	*GTE Buffer (50 mM Glucose, 25 mM Tris, 10 mM EDTA)		*GTE Buffer (50 mM Glucose, 25 mM Tris, 10 mM EDTA)
	*Lysis Solution (0.2N NaOH, 1% SDS)		*Lysis Solution (0.2N NaOH, 1% SDS)
	*Sodium Acetate (pH 4.8 and 3M pH 7)		*Sodium Acetate (3M pH 4.8 and 3M pH 7; pH with Acetic Acid)
	*Ultracentrifuge tubes (Beckman 362185)		*Ultracentrifuge tubes (Beckman 362185)
	*Water-Saturated N-butanol		*Water-Saturated N-butanol

Davebrid: wrote initial page

2009-05-27T14:24:17Z

wrote initial page

New page

==Materials==
*GTE Buffer (50 mM Glucose, 25 mM Tris, 10 mM EDTA)
*Lysis Solution (0.2N NaOH, 1% SDS)
*Sodium Acetate (pH 4.8 and 3M pH 7)
*Ultracentrifuge tubes (Beckman 362185)
*Water-Saturated N-butanol
*Absolute ethanol

Protocol
#Grow 1L overnight culture in 2XYT media with antibiotic
#Pellet and resuspend in 20 mL of GTE buffer
#Add 40 mL Lysis Solution for 5 minutes
#Add 30 mL Sodium Acetate pH 4.8 for 10 minutes
#Spin in JA-10 for 20 min at 8000 RPM
#Remove supernatant and add 0.7 vol Isopropanol
#Spin 20 min at 8000 RPM
#Resuspend DNA in 6 mL of distilled water. Add 10g of CsCl and dissolve.
#Load into two ultracentrifuge tubes then add 25 uL EtBR to each tube
#Centrifuge 60 000 RPM (250 000g) O/N in NVT90 rotor
#Withdraw the upper band with a 18g needle and reload into 1g/L CsCl for second spin
#Remove band and extract EtBr with butanol until clear
#Dialyse overnight in 2L of TE
#Precipitate with 3 vol absolute ethanol and 1/10 vol of 3M Acetate pH 7
#Resuspend to a final concentration of 5-10 mg/mL in TE