PCR Problems

Richard K. Meister rmeister at POP.SERVICE.OHIO-STATE.EDU
Wed Jan 29 12:19:36 EST 1997


Greetings, all!

We have had trouble with our PCR results for over a month, and all of our
diagnosis efforts have been confounded.  Perhaps one or more of you with a
fresh perspective can be of help.

We are using 21-mer primers (that have been re-made 3 times, by two
different oligo suppliers) to amplify a 290 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 60 C ( 5 degrees below the Tm of the primers)
under standard salt conditions.

We 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.

We would appreciate any ideas as to what the source of our problem might be.
Replies may be sent to the list or directly to Dr. Kathleen Hayes at
Hayes.9 at osu.edu.

Thanks in advance.
.
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*  Richard K. Meister                  Email:  Meister.1 at osu.edu  *
*  The Ohio State University           Voice:  (614) 292-9716     *
*  Dept. of Veterinary Biosciences     FAX:    (614) 292-6473     *
*  Cytometry Instrumentation Lab                                  *
*  1925 Coffey Road                                               *
*  Columbus, OH  43210  U.S.A.                                    *
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