RT-PCR question

Jim Owens jow at helix.nih.gov
Wed Dec 22 16:34:08 EST 1993


In article <CIGC12.DBq at news.cis.umn.edu> Michael Man,
mman at molbio.cbs.umn.edu writes:
>>Sure enough, there were cDNAs from both strands.  This was not
unexpected
>>since one of the genes is known to be transcribed with different splice
>>sites from both strands.  However, when the cDNAs amplified by PCR are
>                                                     ^^^^^^^^^^^^^^^^^
>What are the primers used? How big is the PCR product?  Can you design
>PCR primers to cover broader range?
>
>>sequenced the same two splice variants appear no matter which primer is
>>used!  One variant has 37 or 38 bases inserted from out of nowhere at
the
>>junction between the two chromosomes.

PCR products range from 200 - 1100 bp depending on the primer pair used. 
In other words, I have five primers (beginning in an exon) for one strand
and six (some within an exon and some from the distal intron) for the
other strand, and I have sequenced both strands of more than one PCR
product for each variant.  The primers range in size from 23 to 25 bases
with no hairpins or 3' sequences that would anneal to each other or
misprime in the sequences in question according to Amplify 1.2 for the
Macintosh.

The thought has crossed my mind that there might be only one strand
transcribed and that I have somehow contaminated one or more reagents
with them.  To prevent this, I use "aerosol resistant" tips only once.   
But I may need to change pipettors?  Is there a good way to demonstrate
that this is or is not the case?

Thanks,

Jim Owens



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