Why low second-strand cDNA?

James Graham graham at biodec.wustl.edu
Wed Apr 24 17:57:18 EST 1996


Comments welcome on the possible causes of low conversion of 
good first strand cDNA synthesis to double stranded product.

Of course, the second strand is primed by RNA following RNAse H
cleavage, and may have been degraded during a 1 h first strand 
synthesis. Is there a convenient way to rescue this first strand 
material (eg. random prime and fill?). Short products are not 
of conern, but lack of linker-ligatable product ends certainly is.

Does anyone add additional random primers during the second-strand 
synthesis? Is there a problem with this other than reduced 
length that I've overlooked? :)

Thanks much,

Jim
J. Graham PhD 



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