I have already tried decreasing primer concentration, Touch-down PCR and increasing annealing temperature. My pcr product is of 200bp. Gel image of my pcr product with two different dna samples, it is touch-down pcr.
How specific is your primer? Are you trying to do PCR with degenerate primers? The size of the product you mentioned is less and thus it is relatively easy to get if you are using specific primer sets. Furthermore, you should decrease the Mg concentration as Alejandro mentioned earlier. How much DNA are you using? I think you can dilute the DNA before using.
But all things considered it is much better to design a new primer pair (if possible).
Hope it helps.
Did you test your primers on PrimerBlast?
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
Introduce the sequence of both primers in "Primer Parameters"
On "Primer Pair Specificity Checking Parameters", Change the Database to "nr" and in Organism delete "Homo Sapiens" and leave it blank, then press get primers and wait for the results, it's supposed that your sequence of interest will be BLASTED (showed) with the specific product size; however, it's possible that the primers are also matching another region in the genome of your organism or in other cases the host.
Furthermore, did you try decreasing your Mg concentration? The less Mg concentration in your PCR reaction, the more stringent will be your reaction. For that purpose, you can use a GOTAQ Flexi Mastermix where you can add the Mg and you can do it by 0.2 mM increments.
Are you sure your template is not degraded? Did you run the template on gel to check? If your template is intact, then maybe you have to change to a different set of primers with higher annealing temperature.
How specific is your primer? Are you trying to do PCR with degenerate primers? The size of the product you mentioned is less and thus it is relatively easy to get if you are using specific primer sets. Furthermore, you should decrease the Mg concentration as Alejandro mentioned earlier. How much DNA are you using? I think you can dilute the DNA before using.
But all things considered it is much better to design a new primer pair (if possible).
Hope it helps.
The comments already above give a lot of helpful suggestions. To get a more specific answer you should share additional information in addition to the questions already raised. What template are you using? Did you visualize the template as a control?
Dear Nirali,
PCR amplification of high molecular weight DNA (as evident in the attached gel photo) is just not possible using conventional PCR conditions (for amplification of 200 bp) no matter how unspecific the primers may be. Your problem is laying elsewhere. Do you have a gel with markers, PCR blank, and negative control? What was the agarose percentage in the above gel?
@ Alejandro Olmedo & Bodhisattwa Banerjee: Thanks for the suggestions, I have tried decreasing MgCl con but it seems there might be problem with the primers sequence itself. Reconsidering to design new primer set
@Sneha Bairy: DNA is intact as I have use it for other pcr products also.
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