To all and more specifically to Pim and Preston,
I developped a 5' extension protocol during my years in the mammalian
world an fortunately, it was reported in a paper of friends (hi Morrie and
Frabncis) PNAS 263: 17987. I did primer extension few weeks ago using it (ON
annealing at 25 C, in 50% formamide, as for RNase protection) with 100
ug total RNA and, surprise, it worked fine!! One modification. Got rid of the
nucleotide (after labeling the primer) by 2 ammonium acetate precipitation
using glycogen as carrier. Not sure but everything went fine.
Hope it is helpful.
Pierre Belhumeur
Microbiology, U. of Montreal
belhumep at ere.umontreal.ca
