I used the primers (5′- GATGTGAAACACTTTGCTCCT-3′ (sense) and
5′-ATAGCGACACTTGGATAGGGG-3′ (antisense) cited in different literature's for Annx II to have RT-PCR amplification in cancer cell lines including MDAMD 231. I used the PCR or RT-PCR conditions as mentioned in the concerned literatures. cDNA is giving good signal for positive control (GAPDH) always. However, I am unable to amplify Annx II with the above primers. I made gradient PCR to find out the proper Tm. That also failed. I then doubled the template and primer quantity, added DMSO in PCR reaction. GAPDH is getting doubled but no signal I am getting for Annx II. Can any body suggest where could be the problem(s).
something may be wrong with your Annx primers. I would design/order new sets of primers.
Thanks Imam for your answer. That is also I am thinking as alternate. But before that I would like have more inputs as many literature published with this primer set.
Always try to design more pairs of primers, my students always disign at least 5 pairs of primers following the software. you should be fine.
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