missing bands and ghost bands
Brian P Higgins
(by higginsb78 from gmail.com)
Tue Sep 30 09:12:10 EST 2008
One quick question: have any of your coworkers actually tried to run the PCR
that you seem to be having problems with? If they can get it to work well,
then the problem is your technique. No offense, but 8 months with shoddy
PCR results is 7 and a half months too long--someone should have helped you
with this LONG ago. You say that your technique is the only culprit, but
what about template and primers? You didn't define what constituted your
mastermix, so I'm assuming that contains the usual: buffer, Mg, Taq, and
dNTPs. Is it possible you miscalculated your primer dilutions and/or
template concentration? Just a thought.
I agree with Christian--more info would help.
On Tue, Sep 30, 2008 at 5:09 AM, Christian Praetorius <prae from gmx.net> wrote:
> "Manuel Gadin" <manuelg from telus.net> wrote:
> First: Could you please set the length of your line to something like
> 70 characters? That makes it much easier to read.
> >The method/procedure , master mix , gels and thermocycler that I am using
> is the same that my
> >co-lab workers are using. Having said that, my techniques could be the
> only culprit. I am the only
> >one that cannot get consistent results. I am failry new to PCR.Sometimes,
> I would be missing bands
> >in my positive control and also I would get ghost bands. Few of my co
> workers had watched me run
> >the procedure and the only comment I got was maybe the parafilm degraded
> my DNA bands. The
> >parafilm would sometimes stick to my pipet tips when I am mixing the
> samples with the loading dye.
> >Other than that, they cannot offer any solution.
> I don't believe that Parafilm makes any trouble here. I am using the
> same method for years, and when I had problems, it was always
> something different, although it is not always easy to find.
> Please describe your protocol in detail, what is the origin of your
> DNA and so on. And if possible, show a photo of one of your gels.
> X-no-Sig: yes
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