PCR problems of probes

hodges bradley l segdoh at uxa.cso.uiuc.edu
Mon Feb 6 15:24:37 EST 1995

I have tried to use PCR probes for screening libraries and have had no 
success.  You said that your copied PCR products span a wider range of 
molecular weights than your control.  This indicates that you may be 
getting a lot of nonspecific amplification.  Shouldn't you get one 
distinct band after PCRing an isolated fragment?  If not, you may need to 
go over your protocols.  May I suggest using a random priming kit.  We 
now use Stratagenes Prime-It Rmt.  If you feel PCR is your best 
alternative, write me back and I will help you with your protocol.

On 29 Jan 1995, Chris Barry wrote:

> I am having trouble with my probes. Probes given to me hybridize
> perfectly but when I PCR them to make my own, they do not hybridize 
> at all. I dont think it is not a problem with my washings because
> the hybridization always works fine when the controll probes
> are used on the same slide. The only difference I have noticed between
> the controll & PCR copied probes is the band width of the copies
> span a wider range of molecular weights when run on an agarose
> gell. Am I doing something wrong during the amplification procedure?
> How can I remedy the problem?
> 				Confused Undergrad :-(

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