Questions about PCR amplification using cDNA (RT from total RNA). - (Jun/19/2008 )

Hello,

I have problems with PCR using cDNA (RT from total RNA of my cells, using iScript cDNA synthesis kit from Bio-Rad) as template. The PCR product size should be 2.5kb, but I only got non specific bands. I used pfuUltra from Stratagene. I am wondering if it is possible to amplify a 2.5 kb fragment from cDNA reverse transcripted from total RNA. Thanks very much in advance.

if the predicted transcript is that long then you should be able to amplify it. to reduce the number of non specific bands increase the temperature. having second thoughts, why not trying to amplify smaller fragments of the same transcript? should amplify easier

cDNA synthesis may not be giving you enough longer transcripts. have you tried that step with transcript-specific primers?

also, what's the integrity of your total RNA? have you checked it on a gel? antique tech, I know...but if there's any shearing or degradation you will get truncated cDNAs and it will be impossible to PCR the entire fragment

good luck

Amplifying the target in two halves might be a good strategy. The two targets may be easier to isolate than one big one because: a. smaller targets are easier to amplify than larger ones; and b. the cDNA for the smaller targets is more likely to be there than the cDNA for the larger one. It could also be just that your PCR conditions are not good enough. Try a different tissue where your gene is expressed, use more cDNA or if the target is GC-rich use some betaine.

Good luck, Rob

Thank you, toejam, I am trying to amplify the big fragment because I need to clone the gene of my interest (which is that big)into TOPO blunt end cloning vector.

Thanks, aimikins, I have never tried that with transcript-specific primers. My total RNA is good, but my cDNA is synthesized 10 months ago, does it matter?

Thanks, killerkoz17, did you mean amplifying the target in two halves and then ligating them together? How to carry it out? My pfuUltra is too old, it might be a problem, I will try fresh pfu and optimize the conditions.