Long RT-PCR

[Brian Foley]

Kristina Gruden, IJS (Kristina.Gruden at ijs.si) wrote:
: Hi!

: I would like to get a 2,6kb long RT-PCR product. I've succesfully produced
: ... deletion to save space...

	I found it difficult, but not impossible, to amplify the 2.6 kb 
complete cDNA for elongation factor 2 (a relatively highly-expressed 
gene) from mammalian cells.
	The key was getting good RNA.  I had the best luck with selecting
poly-A RNA, but I was also successful using total RNA.  Check that you
have good RNA by running a gel and looking for good high-molecular weight
RNA and no degradation of the rRNA bands if using total RNA.
	Any of the reverse transcriptases worked fine if I had
good RNA.  It was the quality of the RNA that was key.  

: Thanks
: 	Kristina

--
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*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *
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