2 replies to this topic

[jpjp]

Hi,
I am trying to do 5' RACE using cDNA from mouse brain on a transcript with a GC-rich stretch close to the 5' end (77% GC). I am using the RLM RACE kit from ambion, doing the RT with AMV RT and either random decamers or a mix of random decamers and gene specific primers, but I have also tried to amplify from the commercial Clonetech Marathon 5'RACE library. The GC-rich stretch spans most of the 5' end including the first exon, and all the transcripts that I manage to amplify don't start further upstream than beginning of exon 2, so right after the 3' end of the GC stretch.
My guess was that the AMV RT did not manage to reverse transcribe the GC stretch, so I tried to use thermoscript at 65°C. But again I did not manage to find any transcript starting upstream of exon 2.
What else can I try in order to get transcripts starting upstream of exon 2? Data from BAC transgenic mice seem to indicate that these transcripts exist indeed inside the brain...
Thanks for your help.

[phage434]

Have you tried adding some of the PCR enhancers such as betaine to the RT reaction? They help in GC rich PCR, and may help here, though I have no experience to back that up.

[scolix]

Try using DMSO or Betaine for amplifying GC rich sequences.