Difference between revisions of "Cesium Chloride Preparation of DNA"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (added concentration to Acetate Buffer) |
Davebridges (Talk | contribs) (updated cesium concentration) |
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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/ | + | #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 | ||
Latest revision as of 22:03, 17 January 2012
Materials
- GTE Buffer (50 mM Glucose, 25 mM Tris, 10 mM EDTA)
- Lysis Solution (0.2N NaOH, 1% SDS)
- Sodium Acetate (3M pH 4.8 and 3M pH 7; pH with Acetic Acid)
- 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 0.1g/mL 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
