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Looking for explains for procedures in DNA extraction

  1. Jul 14, 2008 #1
    Hi there,
    I tried Google and seems that there's not much information around.
    I'm thinking what are the use of Buffer ATL, AL, AW1, AW2 and AE, and ethanol added to the sample.
    Anyone can help? I'd appreciate even if you post me any reference links:approve:
    Thanks
     
  2. jcsd
  3. Jul 14, 2008 #2

    Moonbear

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    Are those terms you've listed the names of reagents in a kit?

    If you're doing a column separation, generally, you're getting your DNA and other impurities into a solution, prepping the column with the buffer so it's ready for the solution containing DNA, running the DNA solution through the substrate in the column, then washing the column with another solution that releases the DNA from the substrate and lets it pass into your collection vessel. The ethanol lets you precipitate the DNA so you can then dry it as purified DNA.

    I may have missed some steps since it's been ages since I've attempted DNA extraction (others here have much more experience with this), and I've been really general, because I've always just used kits for it, and every brand has its own proprietary solutions to use with it, which have whatever names they gave them. Of course it's good to understand the general concept so you know why each step in the kit is important and when to panic or where you could recover a lost sample.
     
  4. Jul 15, 2008 #3
    Indeed, you need to provide the protocol and/or the buffer compositions to get an answer. There are a lot of different protocols and kits.

    However, I happen to know that these buffers are from QIAGEN kits for tissue DNA extraction. Now for an answer I would like you to read the manual first and then ask more specifically.

    Small hint. The usual procedure for DNA extraction (on columns) are:

    Cell lysis and debris removal

    adsorption of DNA to column under specific conditions (I think it was high salt in QIAGEN columns)

    washing to remove unspecifically bound substrates, including DNA binding proteins
    elution of DNA (under low salt conditions)

    sometimes precipitation and washing of DNA (to remove salts)

    Now think in which order you used the buffers.
     
  5. Jul 15, 2008 #4

    Andy Resnick

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    I'm doing some this exact procedure now, so I've been happy to read the responses. But, I've noticed that some kits are much more complicated than others- my mRNA extraction kit (cell suspension) involves a small table-top centrifuge and takes 1 hour (if that), my miniprep (plasmid extraction from E. Coli) is also quick and simple, but my MaxiPrep kit (a scaled-up miniprep, as far as I can tell), involves quite a bit of equipment (chilled ultracentrifuge, for example), and a lot more buffer solutions.

    What's up with that?
     
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