>100% 2nd-strand cDNA ("snapback")?

[Jim Graham]

Greetings,

Anyone one else get routinely greater than 100% (eg. 500%) radioactively 
traced second strand yield when synthesizing cDNA on control templates 
(transcripts). I'm using the MS2 genome and an RNAseH, DNA pol I 
procedure at 14C for 3 hours following random primed first strand (about 
15-20% 1st strand conversion).

My GC rich sample RNA has the opposite problem, 20-30% second strand 
converions at best. How can I imporve second strand synthesis without 
the dread "snappback" synthesis by pol I? 

Please send me copies of all posts. Thanks

Regards,

Jim
J. Graham PhD
Biology Department