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.
Help!! I have been trying to get consistent results for 8 monthss but to no avail!!!
"Manuel Gadin" <[Only registered users see links. ]> wrote:
First: Could you please set the length of your line to something like
70 characters? That makes it much easier to read.
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.
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 <[Only registered users see links. ]> wrote: