purify PCR products using Wizard Prep (Promega)

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Thu Jul 7 10:27:42 EST 1994


 In article <2ush9n$mnp at agate.berkeley.edu>
 lab_winoto at maillink.berkeley.edu. (Winoto lab) writes:

>> Finally, people often get acceptable yields using straight physical methods
>> for DNA from gel purification, such as freeze and squeeze, etc. You might want
>> to check out a very quick and dirty method in BioTechniques (sometime this
>> year) in which the gel slice is simply pushed through a 0.45 micron syringe
>> filter.
>> 
>> Daniel Kim

> Dan,
>
> I've tried the squeeze method you mentioned and was never able to get
> it to work that well.  Have you tried it?  What kind of results did you
> get?
>
> Pendragon 

On Mon, 9 May 1994, I posted a comparison of different methods for this.
Here's a quick recap...

|                    ...I've tested various methods for recovery of DNA by
| enbedding 20 ul of 100 ng/ul BRL 1 kb DNA ladder in 250 ul of 1.0 % agarose
| within a microtitre well. After cooling, the plug was removed and DNA
| extracted by various techniques. The recovered DNA was then run on another
| agarose gel and compared to the equivalent amount of ladder DNA by the eyeball
| (no relation to Dr. "eyeball") method when stained with ethidium bromide.
| The results showed that the size of DNA recovered was not affected by any of
| these, ie. the largest bands of the 1 kb ladder looked just fine. The
| comparative yield in decreasing order was:
| 
|          method                                         percent recovery
|          ------                                         ----------------
| Syringe method {Li1993}                                    90-100 %
| Millipore Ultra-free-MC; 20 min. @6000                     60-70  %
| Costar Spin-X LC; 20 min. at 6000 RPM                         60-70  %
| BIO101 Gene-clean; 5 ul of glassmilk                       50-60  %
| Garlic press bought at K-mart w/Whatman paper              30-40  %
| 1.0 ml eppendorf tip w/fluff; 60 sec. at 3000 RPM             20-30  %

The reference for the syringe method is:

@article{Li1993,
author = "Q. Li
     and C. L. Ownby",
title = "A rapid method for extraction of {DNA} from
agarose gels using a syringe",
journal = "BioTechniques",
volume = "15",
pages = "976-978",
year = "1993"}

But, this method simply uses a syringe without a 0.45 micron filter attached.

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
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