random primed cDNA: is there a lenght limit? - (Dec/03/2008 )

Hi everyone,
I am working on cDNA synthesis of repeated DNA in mammals. For this I am using random primed cDNA first strand reaction. Everything is going fine (No DNA contamination, Housekeeping RT controls). I am trying to amplify by RT a fragment of 800 bp of one specific sequence, and at the moment no amplification I get. For a small part of 150 bp of the sama sequence, the RT worked. I am asking whether there is a limit for the lenght of the amplification when you were using random primer for the synthesis of the cDNA. Theoretically it should be not a problem, but well, I don't know.
Anyone has amplified by RT more than 1 kb from random primed cDNA?
regards

German

German[/quote]
I don't know of any article for this - but random primed cDNA has given me 800bp PCR products. Theoratically it should be more and more difficult as your expected product size increases.

I would go for oligodt priming if this remains a problem.

You mentioned repeated DNA, you meant repeat sequence? that may be a problem too, for RT as well as PCR.
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Thanks for the reply Cell Counter.
I will try once more with other sequences of similar size. Concerning oligo dT, I am not sure if the repeated DNA I am working with has a poly A tail, this kind of sequences don't code for mRNA.
anyway I will make also a new cDNA using oligo dT, let's see, but I bet it will not work
this repeated DNA I am able to amplify it by PCR from genomic DNA without problems, you only have to manage the amount of sample and polymerase, two very critical factors.
regards

If you know the sequence already, you can also use gene specific primers. Depends upon your exact project though.