High background on Northern Blot

GUT at QUCDN.QueensU.CA GUT at QUCDN.QueensU.CA
Wed Sep 21 19:19:10 EST 1994


Dear netters,

I wish to get some advice from any one with experience in performing Northern h
ybridization. Briefly, I transferred total RNA onto the Nytran nylon membrane a
nd probed the mRNA with a fluorescein-dUTP-labeled plasmid(pUC19 plus a 1.1 kb
insert). The hybridization protocol followed the instruction manual from Dupont
, supplier of the nonradioactive nucleic acid detection kit. Essentially, the
hybridization buffer contains 0.25 M sodium phosphate (pH 7.2), 5% SDS, 1 mM ED
TA and 0.5% blocking reagent supplied by Dupont. The detection system seems to
be a much better alternative than the radioactive approach. A clear signal was
seen after 5 min exposure of the film. However, the background of the autoradio
graph was very high. I seems that my plasmid probe also crosshybridized to the
28S ribosomal RNA. Several factors may be responsible for the prblem. One possi
bility is that I used the whole plasmid as a template in probe labelling. I als
o heard that nylon membrane usually generates more noise than nitrocellulose
membrane. Finally, I am wondering if I should isolate poly A mRNA to avoid the
nonspecific hybridization problem. Any suggestion will be greatly appreciated.

T. Andrew Gu
Biology Department, queen's University, Kingston, Ontario, CANADA
e-mail: gut at qucdn.queensu.ca




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