Hi, What could be the reason, gene of my interest could not be amplified even, I am running optimized PCR conditions and universal primers to target gene of my interest to amplify.
Hi ..
At this point of time there can be many reasons...and you have to check them one by one...if you have done PCR earlier, keep that product as a positive control to see if everything in the PCR mix is working fine or not.....If this looks fine then for a new pair of primer...you may have to check only few parameters,....quality of template, primer conc., annealing temperature, PCR program etc....
.if you can elaborate a little bit whether you have attempted some more optimizations...the problem can be easily sorted out...
Hi,
There could be many reasons !!! Without describing what you exactly performed and what results you obtained, please do not expect any good suggestions from anyone !!!! Well, in most of the PCR reactions, there can be some minor differences in protocol you refer and the conditions that actually work for you. That is because you use different reagents, different lab conditions, different instruments that that of the original. You might have to adjust some parameters. This is just a more generalized scenario .... there could be many things going inside !!!!!
Hi,
Few questions to ask? Firstly, did u managed to get some results (Amplification bands) when using the same conditions and the primers before? Are the conditions optimized for the particular primers and gene? In the first case, if in case u have obtained the result before, but now u r unable, then u have to check the DNA template. Because, the DNA might have degraded into fragments and hence your amplification is not occurred. The primer binding site is no more there to initiate the amplification. You cannot just judge the quality of DNA based on quantification result because the concentration and purity still will be high though the DNA has been degraded. So, just get a freshly extracted DNA and do the PCR. In the second case, do the optimization again for the particular primers if in case the conditions used are not for the respective primers. Lastly, do check your storage condition of ur primers as well.. Hope these will helpful for u..Gud luck..
Thanks Thanes, your points are valid, I ll do the same as you suggested. Thanks.
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