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Primer Extension

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#1 anonymous



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Posted 09 February 2001 - 10:00 PM

I am trying to do Primer Extension experiment on two different genes. I have good primers set at about +100, and they both work fine for sequencing. The problem comes when I try the Reverse Transcription part of the protocol. I have yet to see a band in the right place. I do see a smear from about 10 to 80 bp, with an especially intense spot that runs even with bands of about 70 bp. This anomalous smear appears even in the absence of RNA or primer. This smear disappears if I ethanol precipitate the cDNA before running the gel. I have a kit from Promega, which comes with control RNA and a primer for it. I get the same results with this RNA. I think the smear and the lack of a band are connected, but I can't explain either one, and neither can the tech support people at Promege. Does anyone out ther have any suggestions? Thanks.

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