RT-PCR - (May/20/2009 )

Hi everyone

I have a small oligo primer of 20 mer and the PCR product size should be at 158bp and i am only getting bands at 1.5kb band on gel electrophoresis which is too large!! My oligo primers (F and R) have the same melting temperature at 62'C so i used an annealing temperature equal to the melting temperature for 28 cycles. Can anyone help me on how to calculate the required temperatures and how to programme my elongation?????????????

Can you please send me a link to help me with PCR ??????????

thanx in advance.

Hi!

Since you are using an annealing temperature equal to the Tm of your primers (which should be the highest one you should be able to use for the PCR to work), I think that the problem is not there.

However, why don't you try to put a shorter extension time in your PCR program? If your desired band has 158 bp, an extension step of 20 seconds should be more than enough, and fragments as big as 1.5 kb will not be amplified. (I normally calculate an extension step of 1 minute for 1000 bp, 30 seconds for 500 bp, and so on)

The PCR program I would use is:
1) 94 ºC, 2 min.
2) 94 ºC, 30 sec.
3) 62 ºC, 30 sec. (if this temperature works fine for you)
4) 72 ºC, 20 sec.
5) Go to step 2) 40 times (or less, you don't need to put so many cycles)
6) 72 ºC, 10 min
7) 4 ºC .....

I hope I've been useful. Good luck!

i would try a lower annealing tempearture than the one that u r using, maybe 57-59C. again, lot of variables crop up in a PCR, depending on the GC/AT richness of the template that u r using, the polymerase that u r using etc. Unless u mention about these factors, difficult to suggest the suitable parameters..

Hi!

Usually your annealing temperature should be a few degrees below your melting temperature, so try it 3° to 5° C lower.
Another question: What kind of template do you use? Genomic DNA, copy DNA? Are these intron spanning primers?


All the best!