Problem of Smear in PCR - Getting Smear in negetive control (Jun/05/2006 )
I have had trouble with our PCR results for over a month, and all of my
diagnosis efforts have been confounded. Perhaps one or more of you with a
fresh perspective can be of help.
i am using 26-mer primers to amplify a 2.9 kb sequence of the target DNA.
These primer sequences were carefully designed and have worked fine in
the past. The problem is that recently, when running the products out on a
1% agarose gel, we get a smear that is generally larger (3 - 10 kb) than the
anticipated product. This smear even appears in the reaction mix control
(all reagents except template). The smear persists even when the annealing
temperature is dropped to 57 C ( 5 degrees below the Tm of the primers)
under standard salt conditions.
i have tried using all new reagents and plasticware. All non-commercially
supplied reagents were made fresh with sterile "for injection" water.
Reactions were prepared in a clean room in a static hood, and positive
displacement pipettors were used.
I would appreciate any ideas as to what the source of our problem might be.
Replies may be sent to the list or directly to akhil_iabt@rediffmail.com
your commercially supplied reagents may be contaminated.
your sterile water may be contaminated. sterile water may contain dead organisms which could contribute dna to your samples.
your non-supplied reagents (presuspension or dilution) may be contaminated.
ones, an student here had the same problem, he changed everything... until he discovered his primers were contaminated!!
thanks Aztecan princess
but how can i find out my primers r contaminated, bcoz if i set rection without primers definitely it ll not show smear as primer is imp for any kind of amplification.
have you tried re-ordering your primers? perhaps they have degraded in the freezer and you are now getting non-specific amplification from truncated primer bits