"LONG" RT/PCR

[Elizabeth Estabrook]

Dear Steve

Regarding RT/PCR of BYMV

You say that you suspect that the RT is the limiting step but have you 
actually checked your RT reaction.

You could check to see if you are getting a cDNA of proper length by 
labelling you RT step and checking the product on an alkaline agarose gel.
So if you checked you could be sure that RT was or wasn't working.

My two cents worth is that the RT step is *usually* the most difficult 
and that it depends upon several things: RNA purity, integrity and amount;
primer annealing. I do prefer MMLV RT but I don't think that the 
Superscript is necessary for RT/PCR.  I don't know about increasing time 
though, I usually only do 1 hour max, however I have not had the 
opportunity to make RT/PCR on anything bigger than 2.5 kb.

If you would like my specific conditions or further help just let me know

Elizabeth
emestabrook at ucdavis.edu

 Steve Wylie 
(wylie at CSUVAX1.MURDOCH.EDU.AU) wrote:
: Has anyone tried RT/PCR for RNA of 10KB or longer? I have tried to do 
: this with the potyvirus (BYMV) I work with, but so far no success. 4Kb 
: isn't a problem but anything longer is! I suspect the RT to be the 
: limiting step so have tried M-MLV and AMV RT's, incubating for 3-4 hours 
: but still nothing. Any comments will be gratefully accepted.
: Steve Wylie, Murdoch University, Western Australia