Dear Rohit Rohit have you checked the quantity of DNA? Probably increasing it you can obtain better results. Other question, have you tried before these fade primers?

I can think of three reasons:

1) The primers may be inefficiently binding to the target site. This could be due due to SNPs or an improper annealing temp.

2) Do you know the sequence of the amplicon? If so, make sure it is not AT-rich. If it is, then lower extension to 65˚C

3) DNA quality is low. You may have inhibitors in your DNA. Try running a primer set that works as a positive control

Apart from above suggestions, you need to ensure purity of primers, DNA, enzymes, buffer and water. Any contamination including DNAs activity may change your result from one batch to another batch. Try to use freshly prepared each component of PCR reaction including autoclaved water. Also try to quantify genomic DNA precisely and and use in appropriate concentration. Hopefully, you will resolve you problems.

Hello,

I believe that as dr. Narendra Kumar Singh suggested there is an issue with the component freshness/preparation.

Could you share with us the mixture you are preparing? In that way we could address the problem and clarify the doubts.

I would suggest you to freshly prepare the component and change them one by one.

Best regards

For the first, please, provide photo of some working SSRs if you have to compare and electrophoresis conditions. As for me, you should exclude the sourse of the problem: PCR-components, PCR-conditions of just separation in agarose gel (buffer type, componennts, loading buffer (glycerol based is bad) or electrophoresis conditions, oversalted buffer can also cause smeare, etc).

Hi,

have you used new dNTPs stock or are you using an old one? Which concentration are you using for your dNTPs (10 mM would be my guess).

Have you tried to blast the primers, is this your expected size?

Use always fresh dNTPs since freezing/thawing could decrease the efficacy.

As dr. Yaroslav Ivanovych suggested, try to exclude the source of the problem one by one. I would start with new dNTPs, new buffer, new primers, new polymerase and so on.

As dr. Ricardo Julian Licea-Moreno suggested, have you quantified your DNA concentration (nanodrop/lambda DNA or other methods) ? I would suggest to dilute the DNA concentration 10x and try it again.

Best regards