Joyce: DNA precipitation: Difference between revisions
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*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 | *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 | *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 | ||
*Most people seem to use 70% EtOH and 2 volumes of this (although it has been commented that the volume of the wash isn't so important, so 300 ul is probably sufficient) | *Most people seem to use 70% EtOH and 2 volumes of this (although it has been commented that the volume of the wash isn't so important, so 300 ul is probably sufficient) | ||
Latest revision as of 22:34, 10 September 2009
Concept
- DNA needs to be precipitated to remove residual salts that may inhibit other reactions (like restriction digestion, PCR, and so on)
Procedure
- Add enough 3M NaOAc pH 5.2 to make the final volume 0.33 M, vortex
- Add 2 volumes of 99% EtOH, vortex
- 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
- To make the final volume of NaOAc 0.33 M, use 1/10th of the final volume. For instance, if I have 90 ul of a DNA solution, use 10 ul of the 3 M NaOAc to make a final concentration of 0.33 M.
- NaCl apparently helps precipitate SDS better than NaOAc, so use NaCl (final concentration of 0.2 M) when you have a DNA solution that's dirty with SDS. Also check out the Bitesize Bio link to see what salts are better suited to precipitate what DNA.
- Placing the solution at -70°C for a longer time will help precipitate smaller (less than 100 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
- Most people seem to use 70% EtOH and 2 volumes of this (although it has been commented that the volume of the wash isn't so important, so 300 ul is probably sufficient)