Hi,
I have been trying to reverse transcribe total bacterial RNA by polyadenylation and oligod(T) primers... I used the polyA polymerase kit from Epicentre, cleaned up the reaction on spin columns and did the RT with both Ambion and Invitrogen kits... afterwards I did a PCR for some genes and all I get is a smear near the wells, while I don't have any problem when I do the RT with gene specific primers... I ran the RNA on the gel after poly(A) reactions and the rRNA bands show lower migration, their size is increased, so I guess the polyA reaction is not the problem...
Any suggestions?
thanks!
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.
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