DNA Precipitation

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(Procedure)
(Procedure)
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# (Optional) Add 1 ml of 70% EtOH to the pellet and let sit for 5 minutes.
# (Optional) Add 1 ml of 70% EtOH to the pellet and let sit for 5 minutes.
# (Optional) Centrifuge the sample at maximum spped for 5 minutes.
# (Optional) Centrifuge the sample at maximum spped for 5 minutes.
-
# (Optional)Decant and Discard the supernatant.
+
# (Optional) Decant and Discard the supernatant.
# Air-dry the pellet for 10-15 minutes at room temperature until all liquid is gone.  
# Air-dry the pellet for 10-15 minutes at room temperature until all liquid is gone.  
# Resuspend in desired volume of water or buffer
# Resuspend in desired volume of water or buffer

Revision as of 15:22, 10 June 2009

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Contents

Overview

This protocol can be used to concentrate DNA, or to change the buffer the DNA is suspended in. It can also be coupled with phenol chloroform extraction for the purifying nucleic acids. This protocol also works for RNA precipitation (take care to use RNAse free materials in this case).

Materials

  • 3M NaOAc pH 5.2
  • EtOH 95%
  • Glycogen (optional)

Procedure

  1. Add 0.1 volumes of 3M Sodium Acetate solution to 1 volume of DNA sample.
  2. Add 1ul Glycogen to the DNA sample.
  3. Add 2 volumes of 95% EtOH to the DNA Sample.
  4. Store the solution overnight at -20°C or for 30 minutes at -80°C.
  5. Centrifuge the solution at maximum speed for least 15 minutes.
  6. Decant and discard the supernatant.
  7. (Optional) Add 1 ml of 70% EtOH to the pellet and let sit for 5 minutes.
  8. (Optional) Centrifuge the sample at maximum spped for 5 minutes.
  9. (Optional) Decant and Discard the supernatant.
  10. Air-dry the pellet for 10-15 minutes at room temperature until all liquid is gone.
  11. Resuspend in desired volume of water or buffer

Notes

-20°C for an hour is fine for using larger (1mL of bacterial culture, plasmid) amounts of DNA

The DNA pellet will not always be visible depending on how much DNA you are precipitating. So always take care in loading your samples in the centrifuge to remember the direction they are facing. The DNA pellet will be on the part of the tube facing the outside of the centrifuge.

This protocol will precipitate all nucleic acids, not just DNA. If you do not want RNA in your sample, one of the many ways to deal with it is to simply resuspend in TE + RNAse at the last step and leave it at room temperature for 15mins-1hr.

References

Links

Contact

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