pcr problem

[vageeshwari]

pcr products are uncertain. one day the bands are present, other day its weak,and one day it is missing. i am mixing the ingradients nicely, ive checked primer,enzyme, buffer ,dntp ,water and dna are are effiicient but the results are uncertain

[jiajia1987]

why don't you describe your problem to us in detail and perhaps let us know about the parameters and even include a gel picture so we can help you better?

[lala0453]

Wow! I’ve never heard of this before and I think they’re awesome!

[aje00325]

Valuable information.

[rumaisya]

the same thing happend to me vageeshwari...

[rumaisya]

and i also still searching for the answer..

[pselva7]

I too faced the same kind of problem vageeshwari, i didnt get any answer still, but keep on trying with adjusting all contents.........

[Aga]

Guys, let us know some more details because PCR reactions you perform are different.

Let's say we can rule out all the problems with reagents - we can suppose they are reliable and new. We can also suppose that you optimised the reaction and use the same temperature and time regime you set for PCR.

By 'uncertain' results I understand your reactions are not reproducible.
Uncertain results may be caused by the quality of your template, contamination, pippettes and tips you're using (this relates to the source of possible contamination or errors in pippetting).
I can only assume that, because I don't know whether you tried asses inter- and intra-assay variations repeating PCR with the same set of reagents and in the same conditions with the same template, for example in triplicates, and still get different uncertain results.

So if you still need answers, could you possibly give us some more information? I guess there are some people that may help you.

[peggy88]

i faced to same problem, i had checked all the parameters such as buffer, thermal cycle, the content of DNA template and etc. the interest gene that i tried to amplified is rs165656.

[pselva7]

hi guys....
At the time when i am starting to amplify a gene in the presence of biased dNTPs i got very little amplification by using takara Taq polymerase, then unfortunately i using another company(Beams biotechnology) Taq polymerase with the same parameters, but this time i didnt get even a less amplification, so i changed every constitutents including pcr buffer, dNTPs, template, chclic condition and aneealing temperatures, but i didnt get any resuults further more. The desired is gene is 76% GC rich region, so we always use small % of formamide. When i using Takara taq polymerase i use 10% formamide still i got amplification. Where as when i start to another taq polymerase i didnt get any results, then finnally i increase the formamide concentration but no positive results. then i reduce the formamide concentration, but this time i got very good amplification, this time i was used 5% formamide. The thing is PCR additives also have crucial role in the amplification of desired gene. So, i recommend you, just think about the additives it may helpful to find a amplification.

Good luck...

Thanks.....

By
Selvam.