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RT-PCR amplification problem


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#1 swatisubodh

swatisubodh

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Posted 21 December 2003 - 11:53 PM

I am working on a dsRNA virus. since secondary structure pose a problem for large fragment amplifications, I am amplifying a 3000bp gene as overlapping fragments. The sequence of the forward primer of one fragment and the reverse primer of the adjacent fragment are complementary sequences, thus forward and reverse primers of adjacent fragments are of opposite polarity. My problem my middle fragment is not amplifying although its flanking fragments have amplified and been sequenced. Since the primers are amplifying adjoining fragments, the primer annealing etc. should not be a problem. What do you suggest? How can I modify my RT-PCR conditions?

#2 go go fly

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Posted 25 December 2003 - 12:17 AM

I am working on a dsRNA virus. since secondary structure pose a problem for large fragment amplifications, I am amplifying a 3000bp gene as overlapping fragments. The  sequence of the forward primer of one fragment and the reverse primer of the adjacent fragment are complementary sequences, thus forward and reverse primers of adjacent fragments are of opposite polarity. My problem my middle fragment is not amplifying although its flanking fragments have amplified and been sequenced. Since the primers are amplifying adjoining fragments, the primer annealing etc. should not be a problem. What do you suggest? How can I modify my RT-PCR conditions?

Hi
I think U could try to amplify small fragment like 1500bp and then reconstruct it. :D




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