just i've been working on PCR these days, and i've repeatedly met a problem that's realy confusing me.
i tried to amplify a cDNA template(345.8ng/ul, OD 260/280=1.6215) got from my myxofibrosarcoma cell line by GAPDH primers, but the only thing i got is a smear in a discrete region with a faint band at the right product size located at the top of the smear. i've checked the concentrations of all my reagents and make sure that they are all in the right conditions and the enzyme was the last one to be added to the master mixture.
here's my protocal:
10X gen taq buffer: 1.5 λ
2.5mM dNTP: 1.5λ
10mM GAPDH primer: F 0.5λ
cDNA template (150μg/λ):1λ
2U Taq: 0.3λ
Total volume: 15λ
(for 35 cycles)
agarose gel used: 2% agarose gel (electrophorese in TBE solution)
and the PCR product is expected to be around 200 bp, but the smear region extends to about 100 bp, and nothing showed up beyond this region. i wonder was it caused because once i sterred my template with tip gently and tried to pipet it two to three times?
please give me some advise to solve the problem, thanks a lot!
a beginner in the molecular biology lab
Edited by uglycell, 20 September 2009 - 08:59 PM.