[Q] How to purify small PCR frg. ?

Julian Parkhill J.Parkhill at bham.ac.uk
Wed Jul 19 11:51:17 EST 1995


In article <3uidfs$4cs at mserv1.dl.ac.uk>, p_rudolph at MORPH.SPACENET.DE
(Peter Rudolph) wrote:

> Hi netters,
> 
> recently a coworker of mine ran into a problem:
> She created a library with PCR using randomized primers,
> then she digested the fragment and want to ligate it into a vector.
> The complete PCR fragment is 96bp, after digestion she gets
> two fragments: 29 and 67bp. She separated them by gel
> electrophoresis on a 2.5% AG.
> The problem is now: How to purify the bigger fragment from the
> gel slice with a really *high* yield?
> She tried freeze-and-squeeze and Qiagen-gel-ex but got
> only very low yields.

Very small fragments of DNA (and up to 1.5kb) are easily eluted from
acrylamide gels.
Run the fragments down a 6% (29:1) acrylamide gel, cut out band, crush in
an eppendorf, add TE and shake at 37 degrees C overnight. Spin out the
acrylamide and extract the s/n with phenol and chloroform then ppt with
sodium acetate and propan-2-ol.

For even greater efficiency use Earl Ruley Elution buffer in place of TE.

EREB: 500mM ammonium acetate, 10mM magnesium acetate, 0.1 percent SDS,
0.1mM EDTA.

I have acheived effectively 100 percent recovery with this protocol, and I
find the resultant DNA is far cleaner and easier to ligate than DNA from
agarose.

good luck,

-- 
          Julian Parkhill   -   J.Parkhill at bham.ac.uk
   CRC Laboratories, Inst. of Cancer Studies, Medical School,
 University of Birmingham, Edgbaston, Birmingham, B15 2TJ, U.K.
Disclaimer: If I thought anybody gave a damn about my opinions....



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