why my Northern blot do not work?

Hongyan Wang h0w0926 at UNIX.TAMU.EDU
Thu Jun 18 13:19:14 EST 1998


I did my Nothern blot, but it did not have any signals. I know the gene
is highly expressed by previous differential screening of the cDNA
library. I followed the protocal of the Amersham booklet with the Hybond
N+ membrane. That is what I did: transfer the RNA use 20x ssc, briefly
wash with 2xssc, UV crosslink, store in -4C for about one week,
pre-hybidize for 3 days, hybridize for 16 hours, wash with 2xsspe 0.1%
SDS two times @ r.t. 10min, 1xsspe @ 65C for 15min, 0.1xsspe @ 65C for
about 45min. After I did the RNA transfer I checked gel, there was no
EtBr Stained RNA left on the gel. The Southern blot I did in the same
box for hybridize and wash  worked beautifully. So I really can not
figure out what is wrong. If any one can help me, I will be very
grateful, thanks!

Hongyan Wang
Texas A&M university



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