Richard K. Meister wrote:
> 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.
> .
> * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
> * 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. *
> * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
It looks like you have tried everything you can to
eliminate the possibility of contamination with template
DNA, and you still get a smear with your primers with
or without added template DNA. There are many things that
can go wrong in PCR, but contamination is the most frequent
problem.
If I were you, I would find someone else nearby
at your University who is getting good PCR results
right now, and ask to borrow some of their reagents.
Get a small aliquot, rather than using some and returning
the reagents when you are done. Use their primers and
template with your nucleotides, polymerase etc...
Use your primers and template with their nucletides,
polymerase etc... Use their thermal cycler for one
or both reactions, or another set of parallel
reactions.
TAQ (and other polymerases) can sometimes
cantain bacterial nucleic acids. These may be problems
for some primer pairs but not others.
At any rate. It is very hard to
trouble shoot when it is not working. Thus if
you can cooperate with a group that is getting
good results, it is much quicker to pinpoint the
source of the problem. Don't overlook even the simplest
thing! Perhaps your PCR works great, but their is
something wrong with running your gels. I suspect that
you would have noticed that problem though, because your
molecular weight markers would smear as well as the
PCR product.
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