Dear Sir,
Please find find the attached file what are my primers and their details along with product length, few are previously optimized with human template standard 50ng DNA. Last 7 and 8 primers are optimized not worked yet and 9th one is getting non specific bands. I checked these primers BLAST also everything is fine.
After optimization, I did the polymerization of my subject (Patients DNA) with that annealing temp (which shows in table). not working. While this PCR, I kept one control which is standard DNA it was amplified remaining not working. I observed in gel it might be smear/no product/empty.
So once again I am going to be standardized these primers with different temp. in gradient PCR (applied Biosystem 96 well).
I could request you to give your valuable suggestion for these (which temp can I keep), and shall I use DMSO (for subject samples) for optimization.
Thanks in advance.
Regards,
Yedukondalu
your primers 7. and 9. differ too much in Tm, I would try to find better solution, higher Tm for reverse primers, if Tm of primer is too low and annealing T high, primers with low Tm may not function at all, As for 8. maybe 56C annealing T would be efficient.
Dear Kollati:
I would suggest the following for you to try:
Primer set 1 – Annealing Temp 52-54 ˚C;
Primer set 2 – 52-54 ˚C;
Primer set 3 – 55-58 ˚C;
primer set 4 - 42 ˚C (I will not recommend to use this primer, Annealing temp very low);
Primer set 5 - 50 ˚C;
primer set 6 - 38 ˚C (I will not recommend to use this primer, Annealing temp very low);
primer set 7 – 49 ˚C;
primer set 8 – 50 ˚C, and
Primer set 9 – 47 ˚C
Dear Sudarsono Sir,
Thank you very much sir, I will try with temp. and get back to you Sir.
I hope this temp. will be worked out.
Thanks
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