Polyadenylation+RT-PCR

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Basak Ozturk

Yeah I know... This is why I did polyadenylation beforehand, and it seems to work. It is just the RT that doesn't. So after polyadenylation I would assume the the mRNA does have a polyA tail...

Ah, that is really true.. i think the oligod(T) primers are a mix between 18-20mer oligod(T)'s... so you are right that they may bind to some other place too, but the size of the smear is waaaay higher , about 3000 bp while the product is supposed to be around 700 :((( How else can I check that I am successfully able to create a cDNA pool with oligod(T) primers? Or do you know any other way? -also not random hexamers, they are creating a whole bunch of small cDNA-

Oh man, this is a great idea!! I will definitely check what I can do... thank you sooo much!!

Hhmmm... there is still one thing... Actually though I do my RT reaction with oligodT primers, the PCR primers are gene specific, and work very well if I do the RT reaction with gene specific primers... So I think that is not the reason, but perhaps the RT efficiency is not very good -but I st'll don't understand, people use oligodT primers quite commonly to generate cDNA pools, why shouldn't it work with me?

:((

But the adapter primers idea is great, especially for EST libraries, I guess... I mean perhaps it helps to reduce the amount of truncated cDNA's.

yeah.. I thought of doing that, but I will have to use the RNA for EST sequencing, and I am afraid that I might encounter lots of truncated sequences if I use random hexamers...