PCR Trouble - (Jun/21/2006 )

I am working on a project that requires simple PCR amplification run, but I am having trouble
getting the right bps. My sample should be at ~420 bps but I keep on getting the band at ~250 bps.
I have tried many methods, pFU with Taq or without Taq, Taq alone, different annealing temperature
55 - 72 C, adding DMSO in PCR... etc

The template and primers are all correct, I have double checked them. Everything seems well to me
but I just could not make the amplification happen. Sometimes I only see primer on the gel and nothing else. Primers design is correct we have checked on that many times; I was thinkg about
secondary structure in the template but that is not the case here. I even tried to digest out the part I need and use it as template to avoid secondary structure and still no luck.

The below is my condition and reagent I used. pFU is from invitrogen alone with buffer. dNTP is freshed made so there is no degradation. Annealing temperature was designed for 60 C but I have already tried lower and higher the temperature and still no luck. Extention time was also tried at 1 min, 2 min even up to 4 min and still no good.

item volume (ul)
Pfu BF (10x) 5
dNTP (10 mM) 1
Sin-Xho I fw 2
Sin-BstE2 bw 2
Temp. DNA 1
Pfu poly 0.5
water 38.5

Cycle(s) Time Temperature
1 45 sec 94C (Initial)
30-35 45 sec 94C (Denaturation)
45 sec 60C (Annealing)
1 min 72 C (extention)

1 10 min 72 C (extention)

1 4C (Soak)


If any of you have any suggestion please do respond. Been stuck on this for a really long time now
Thanks in advance.

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mmm Are you sure your template is ok? Is the gene in it? Could you have a (shorter) isoform?

You say you put 1µl DNA template, what is the concentration of your DNA? I have had trouble with my PCR reaction because i overloaded the mix with DNA. It didn't work first so i upgraded the mix with more and more and more DNA. One day i tried to use less and it suddenly worked.

Here is a website that has helped me in the past a lot. It is for Taq...so if you use a proofreading enzyme you have to see if some adjuvants don't interfere.

http://info.med.yale.edu/genetics/ward/tavi/Trblesht.html

I hope this helps a bit.

greetz
Kitti

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The template is correct and yes my gene is in it. I have done many sets of diagnostic digestion to see if the cut sites and the gene still exist and indeed they are there. I have used concentration of DNA template at 1ng/ul, 5 ng/ul, 10 ng/ul, and 50 ng/ul. All of them gave me the same result. I have tried PCR without Taq and it did not work either. Usually lower template concentration will give you a better result. I just don't know what else to try. I used that site before for DMSO, glycerol reference, and still same result

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mmm seems like you really tried everything in the book...

You said you've checked your primers...maybe the company who constructed them made an error? Can you re-order your primers?

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It's also possible you have a mis-priming. Is there any chance that the 3' end of either primer could misprime between your planned priming sites?
What Tm's do you have? How much difference is there between them? If you have too much difference, you will really reduce your efficiency of amplification. I aim for a Tm difference less than 4C, and will add or remove bases to get that.

From what you've said, the insert isn't the issue. The problem must arise somewhere else, and my best bet is in the primers.

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maybe the GC% of you interest is two high in some area.

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