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
>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 at botany.utoronto.ca
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?
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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