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Help! How to get rid of a repeatedly appearing smear in my RT-PCR result!

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2 replies to this topic

#1 uglycell



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Posted 20 September 2009 - 08:56 PM

hi all,
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:
ddH2O: 9.7λ
10X gen taq buffer: 1.5 λ
2.5mM dNTP: 1.5λ
10mM GAPDH primer: F 0.5λ
R 0.5λ
cDNA template (150μg/λ):1λ
2U Taq: 0.3λ
Total volume: 15λ

PCR program:
94℃ 5min

94℃ 30s
60℃ 30s
72℃ 30s
(for 35 cycles)
72℃ 10min
10℃ 10min

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.

#2 zhongmindai



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Posted 20 September 2009 - 11:42 PM

I can only speculate that your PCR may be over-cycled, try to use less template cDNA and/or -5 cycles of your PCR program.
Your PCR components are not clearly listed (e.g. What's the concentration of Mg2+ in 10X gen taq buffer?). As free Mg2+ in the PCR reaction mixture is influenced by the concentration of dNTPs. A free Mg2+ concentration of about 0.75 mM is optional for Taq DNA polymerase. The most frequently used concentration of Mg2+ (Not free Mg2+) and dNTPs are 1.5 mM and 0.2 mM respectively, take account that 0.2 mM dNTPs chelate 0.8 mM Mg2+, the free Mg2+ is 0.7 mM. In your case, the dNTPs concentration is 0.25 mM, which is a bit higher than the most frequently used concentrations. Maybe this is another problem.
By the way, you did not enclose your electrophoresis image, I can only imaging. : )

Edited by zhongmindai, 20 September 2009 - 11:49 PM.

Zhong-Min Dai

#3 uglycell



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Posted 21 September 2009 - 05:51 PM

OH...here's the photo. Thanks! :)
so it might be the relative concentrations between dNTP and Mg2+ are wrong, i see...
but i got the protocol from my seniors and they had used it for years, no such result ever showed upAttached File  nmfh1_oh931_RT.bmp   1012.55KB   354 downloads.

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