Joyce: DNA precipitation: Difference between revisions
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(New page: ==Procedure== #Add enough NaOAc (or equivalent) to make the final volume 0.33 M #Add 2 volumes of 99% EtOH #Place at -70°C for at least 30 minutes #Spin at 14K (?) at 4°C for 30 minutes...) |
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==Procedure== | ==Procedure== | ||
#Add enough NaOAc (or equivalent) to make the final volume 0.33 M | #Add enough 3M NaOAc pH 5.2(or equivalent) to make the final volume 0.33 M | ||
#Add 2 volumes of 99% EtOH | #Add 2 volumes of 99% EtOH | ||
#Place at -70°C for at least 30 minutes | #Place at -70°C for at least 30 minutes | ||
| Line 12: | Line 12: | ||
#Air dry for 20 minutes at 37°C, dessicate or speed vac it | #Air dry for 20 minutes at 37°C, dessicate or speed vac it | ||
#Resuspend in sterile water or TE | #Resuspend in sterile water or TE | ||
==Notes== | ==Notes== | ||
Revision as of 21:47, 10 September 2009
Procedure
- Add enough 3M NaOAc pH 5.2(or equivalent) to make the final volume 0.33 M
- Add 2 volumes of 99% EtOH
- Place at -70°C for at least 30 minutes
- Spin at 14K (?) at 4°C for 30 minutes
- Pipette off supernatant (don't disturb the pellet)
- Wash (gently invert tube 3-4 times) with 300 ul 80% EtOH
- Spin at 14K for 2 minutes
- Pipette off supernant (don't disturb the pellet)
- Spin down residual EtOH and remove by pipetting
- Air dry for 20 minutes at 37°C, dessicate or speed vac it
- Resuspend in sterile water or TE
Notes
- Placing the solution at -70°C for a longer time will help precipitate smaller (less than 500 bp) DNA fragments
- The pellet will stick strongly to the ependorff wall after treatment with 99% EtOH, but will stick less when using 80% EtOH (because DNA is more soluble in water), so be careful when pipetting off the 80% EtOH not to disturb the pellet
- Organic solvents like EtOH can inhibit downstream reactions (some REs are more sensitive to ethanol than others, for instance), so be sure to pipette off as much as possible using a P10-type tip, and allow the ethanol adequate time to evaporate
