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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