Why low second-strand cDNA?

[James Graham]

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