Difficult RT-PCR

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Wed Oct 27 03:36:10 EST 1999


In article <38161183.1FB2C604 at botany.utoronto.ca>, Andrea Gilpin
<gilpin at botany.utoronto.ca> writes
>Dear Everyone:
>
>We are currently facing difficulty getting RT-PCR products.  Our gene
>has a genomic length of 9 kb, in which there are 8 exons making the
>total coding sequence of 3.6 kb.  We can easily get a 1.2 kb cDNA
>spanning exons 1 and 2, and recently, we have also obtained a 2.9 kb
>fragment of cDNA from exon 1 to exon 6.  But we have had tremendous
>difficulty getting the full-length cDNA with the RT-PCR method.  Does
>anyone have any suggestions, recommendations and/or tips.
>
>Thanks very much,
>
>Zhou
>zhou at botany.utoronto.ca
>

Hi Zhou,

Can you successfully amplify exons 6-8 in another RT-PCR? 

Is there any high G/C or problematic secondary structure at that end.

Have you tried adding DMSO to say 5 -10% final and betaine to 1M final?

Are you doing single tube RT-PCR or the 1'st strand reaction first then
taking an aliquot of that and running the PCR. 

I think you need to see if exons 6-8 amplify and how well relative to an
amplification of exons 1-6. 

Could you get away with amplifying the gene in two overlapping parts and
sticking it back together?

Duncan 
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk



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