chimeric race products???
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Thu Sep 23 07:10:04 EST 1999
In article <email@example.com>, alex dobrovic
<adobrovic at medicine.adelaide.edu.au> writes
>We have seen a lot of alternative splicing with RACE. Some of these
>"unrelated sequences" are possibly introns.
>>I¥ve tried to race several genes with the Marathon kit. I do nested PCRs
>>with little or no product in the first round (30 cycles) and many bands
>>and a smear in the second round 30 cycles as well (with recommended
>>temperatures 72 X5/70 X5/68 X20). the bands I¥ve sequenced so far often
>>start with the expected bases but then the sequence "hops" to another -
>>totaly unrelated - sequence.
If you have trace (and I do mean trace) amounts of DNA in your RNA then
it is possible that you are amplifying up from the genomic contamination
and hence introns. You won't need much contamination to pick this up
with RACE given the no. of cycles.
Are you DNAse treating your RNA and if so are you using Mg in your
buffer or Mn? Mn supposedly gives better destruction of the DNA by
cleaving both strands at the same position whilst with Mg you cleave at
different positions leaving overhangs that in theory can re-anneal,
thereby re-generating your genomic template.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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