Subcloning dirty PCR products

Rafa Maldonado Rafael at genetics.med.utah.edu
Thu Aug 25 15:33:43 EST 1994


In article <1994Aug24.231015.1 at mcclb0>
rosas at mcclb0.med.nyu.edu writes:


 
> Now, here come my questions:
> 1. What are the most likely causes for my PCR product not to get into
> the vector??? Incomplete or non-digestion caused by some interferring
> by-product??? Others???

Yes, of course: oligos, dNTP's and so. With respect to the smear,
possibly they are subproducts of your PCR. If you have too much, the
are going to bug your digestion. Try some optimization of the PCR
reaction.

> 2. Is it possible to get ride of such by-products without repeating
> the PCR reaction (I'm short of primers), say by cutting my PCR product
> out of a LM agarose gel???

Yes, you can try some gel-extraction system, using high resolution
agarose and a very good system of recovery from the gel (as quiagen
beads). But I prefer centricon 100. If you need special protocols,
contact me.

> I will really
> appreciate very much any useful suggestion. I know it may take much
> of your priceless time to answer me, but...you know better than I do
> how satisfactory for yourself
>  it is to offer somebody a hand, knowing
> that the other one has no more than a few words to give you
> in return...THANK YOU!!!.
You're wellcome.

Rafa

----------------------------------
Rafael Maldonado
Howard Hughes Medical Insitute
University of Utah
Rafael at genetics.med.utah.edu



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