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Rt-PCR problem !with 2.5 kb gene - (Apr/16/2010 )

Hi all,
Since I am a beginner in Rt-PCR..I have few questions.....

I am trying to do RT-PCR to get the PaFs gene of 2.5 kb.I am using the Finnzymes in which I used the conditions for the cDNA are template DNA...upto 1ug,10mM dNTP1ul
oligo dt primer 1 and RNAse free water .....

10X RT buffer
RT enzyme mix
water

and then I run the PCR by taaking the cDNA of about 5ul .....................and Finally I tried to optimise the temperatures
initial denaturation 98C for 30sec
denaturation 98 for 10sec
Annealing ___50,55.60.65 for 10sec
extension 72 ,40sec
final extension..72 5min
4 hold......for 30cycles............

but,I didnot get any band or smear at different temperatues 50,55,60,65 .

Primers chosen are test primers(31) and Padc4_LIC ( 59 nt)....

Can any one give suggestion please..how to optimise or proceed to get the 2,5 kb product..

Thanks in Advance.

-biology-

I assume you used the phusion DNA polymerase. Although this enzyme is faster than other polymerases 40sec for 2.5kb seems a bit short to me, I would extend this to 1:20min.
second, I would recommend to use less template, 1 Ál should be enough and increase the cycle number to 40.

-tea-test-

tea-test on Apr 16 2010, 07:45 AM said:

I assume you used the phusion DNA polymerase. Although this enzyme is faster than other polymerases 40sec for 2.5kb seems a bit short to me, I would extend this to 1:20min.
second, I would recommend to use less template, 1 Ál should be enough and increase the cycle number to 40.



oh Great,Then I will try with these parameters.Thanks

-biology-

you can also try the GC buffer together with DMSO which is supplied with the polymerase. I have already encountered genes that were only amplified with this buffer but not with the HF.

-tea-test-