Purification of DNA

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Revision as of 03:15, 25 April 2007 by Jakob Suckale (talk | contribs) (Alcohol precipitation: comparison EtOH 2PrOH)
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There are several methods for purifying DNA. The one you choose depends on the nature of your DNA sample and your downstream applications.

Alcohol precipitation

  1. Ethanol precipitation of nucleic acids
  2. Ethanol precipitation of small DNA fragments
  3. Isopropanol Precipitation for PCR Purification
    • use to concentrate DNA from PCR when you have verified that only a single PCR product exists. When performed properly this procedure exceeds the recovery of QIAquick PCR purification columns

2-propanol (isopropanol) precipitation yields less DNA than EtOH precipitation. (In my hands 60% less on average! Jasu) 2-propanol at RT reduces the risk of co-precipitation of salts (which can interfere with downstream experiments). 2-propanol is preferred for large volumes of DNA since less alcohol is required, plus it's faster since it doesn't require a cooling step. Glassy pellets from 2-propanol precipitation are harder to see than fluffy, salt-containing pellets from ethanol precipitation. 2-propanol pellets are also more loosely attached after centrifugation. Take care when decanting.

Filtration / affinity columns

  1. Centrifugal filtration/Nucleic acids
    • for small DNA fragments (~50-200 bp in length)
    • removes proteins, nucleotides and salts
  2. Miniprep
    • for purifying plasmid DNA from E. coli cells
  3. QIAquick PCR purification
    • for typical DNA fragments (> 200 bp in length)