发信人: mjking (银河渡口), 信区: Biology
标 题: Re: dummy questions__how to get small DNA pieces?
发信站: The unknown SPACE (Tue Jun 26 17:04:30 2001), 转信


1. buy enough amount of commercially available herring (or salmon) sperm DNA.
They are dirty cheap. you can get 100mg/$200 probably anywhere.
2. digest your DNA using AluI restriction enzyme. If AluI does not work
well, try other enzymes that recognize 4-nucleotide palingdromic sequence.
You may combine 2 enzymes in one digestion mixture.
3. run a 6% denatured sequencing gel using a thicker spacer set (like 1mm), or
better (maybe) a 1.5% agarose gel to seperate your DNA.
4. Cut out your DNA of ideal size. Of course, a 1-kb ladder will help you
to check sizes of your digestion products.
5. If you are using agarose gel, elute your DNA from the gel into a dialysis
bag electically. If you are using denatured PAGE gel, soak your gel in
proper amount of water O.N. and seperate you DNA using spin columns.
6. ethanol, phenol extraction of your DNA sample, and ethanol precipitation
in -80 C for 1 hour. Dry your sample and resuspend it in proper amount
of desired buffer or water.



【 在 dafa (呆鸟) 的大作中提到: 】
: I need to use small DNA pieces. It should be linear in solution
: so I can see it clear with AFM or TEM. I donot care about the
: base pair sequence. Say, I donot care if it is 100 bp or 600 bp
: but it need to be stable in linear shape.
: I know with PCR I can get some but I need a large amount, at least
: several mg. Will enzyme help? If so, how to control the length of
: the final product? How to purify it? thanks a lot, :)


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