I want to synthesise a 8500bp cDNA but its RNA has multiple loops (overall deltaG= -960 only predicted for 3500bp in clc mainworkbench). How can i synthesize it? Do loops interrupt cDNA synthesis? Can i use 4-5 primer pairs to synthesize it as 1500-2000bp segments and then use overlap PCR to join them?
Thanks.
you can synthesize short segment ( example 1000 bp) and from it design forward primer at last 100 bp (between 900-1000 bp), then use it to synthesize the second segment. By these steps you can synthesize long cDNA.
Loops doesn’t interfere u reaction and if u think so u can keep sample at 55-65ο c before cDNA synthesis. It better to use high capacity RT enzyme to synthesis long cDNA in single reaction.
What you are planning also is good but it traditional and need more time.
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