PCR purification KITS: Best one??????

Koen A.L. De Smet k.desmet at nospam.ic.ac.uk
Mon Aug 18 02:49:02 EST 1997


Mike Lyristis wrote:
> 
> Hello there,
> 
> Can anyone help me... I am at that point. I perform PCR reactions and I
> get one specific product. I have used the Qiagen clean up kit, the
> Geneclean Kit, and I have done a good ol ammonium acetate precipitation.
> What I see is conversion to what presumably is a single stranded form. I
> have repeated the purification and I get the same result consistently. I
> rang the Qiagen reps and they told me that for DNA that has some unique
> characteristic (such as high A+T content), there is some conversion to a
> single stranded form. I am having an immense amount of trouble with it.
> I am also wasting a lot of time with trouble shooting this problem which
> exists for 4 independent PCR reactions.
> 
> Has anyone any suggestions that may be of some help. I do not know what
> to do...
> 
> Many thanks
> Mike L
> Department of Biochemistry and Cell Biology
> Rice University
> Houston, TX,
> USA

I am not sure what causes your problem, but here is an alternative form of PCR product 
purification by PEG precipitation. It is not a kit, so is much cheaper. It can only be 
doen when you have a single PCR band on a gel. I used to sequence from products purified 
this way.


PEG PRECIPITATION OF PCR PRODUCTS:
1.	Take 45 µl of PCR reaction product.
	  Add:	32.5 µl	40 % PEG
		      10 µl	3 M NaAc pH 5.2
		      12.5 µl	H2O
2.	Leave at R/T for 15 minutes.
3.	Spin 15 minutes.
	  Wash with 70% ethanol.
4.	Dry pellet.
	  dissolve in 20 µl H2O.
5.	Check 2.5 µl on an agarose gel.

Make sure you mix all ingredients well at step 1. PEG is very viscous and if not mixed 
properly wit hyuor DNA, it will not precipitate it and you lose it all.


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        Dr Koen A.L. De Smet
        Research Fellow
        Department of Medical Microbiology
        Imperial College Medical School at St Mary's
        Norfolk Place
        London W2 1PG
        Great Britain
                                     
        Tel: (+44)-(0)171-594 3946  
        Fax: (+44)-(0)171-262-6299   
        http://www.sm.ic.ac.uk/medmicro/home.htm
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