Primer problem

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roniadam
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Primer problem

Post by roniadam » Sat Sep 05, 2009 3:10 am

Hi
I designed a specific primers and blast it to amplify my gene. this primer amplifies a gDNA of the gene. I used dreamtaq kit and its protocol but unfortunately the expected product (2054 bp) was not there and what I got is several bands below and above my expected product size.
Then I have used Phusion hihgfidelity kit but the problem is still there no expected product was there.
I went again and checked the primers sequence and it shows a specific sequence for the gene but until now I cannot solve this problem. I also tried to change thermocycling conditions but still the problem exists.
Can anyone help me what to do?

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jyaron
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Post by jyaron » Sat Sep 05, 2009 10:29 pm

Have you checked for contamination in your reagents by using the kits on a previously tested primer that you know gives the right product size? My lab recently had an issue with contamination in our SuperScript mix that was giving us unwanted banding in our gels and it turned out that our primers were fine.

Maybe you could try ordering a primer for a different section of the gene? We usually order 2-3 different primers for any given gene we're testing for.. after all, it is pretty inexpensive.
Experience: Cell Biology, Confocal Microscopy, Developmental Biology, Evolutionary Biology, Genetics, Genomics, Physiology

roniadam
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Post by roniadam » Tue Sep 08, 2009 3:03 am

Hi Jyaron:

There is no contamination in our reagents as this was excluded by using different primers which gave expected products except with using this pair. I reordered again the primers but the problem still is there :(

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