riboprobes for northern blots
mg690769 at bcm.tmc.edu
Tue May 23 09:18:35 EST 1995
I am trying to use antisense riboprobes for northern blots, and have
come up with major backround problems- nomely hybridization to 28S RNA.
I am using the pCRII vector from invitrogen to transcribe off the T7
promoter, as well as bluescript using the same promoter. Riboprobes range
in size from 300-500 bases and are biased to the 3'UTR.
The labelling reaction used 0.5ug template DNA
with 3ul 32P-ATP (800Ci/mmol)
I tried cleaning it up with EtOH ppt. and used the whole RXN in 10 mls
hybridization solution. My Counter read 600,000 counts/ul, and I had
50ul of labelled probe.
I washed the blots with 2xSSC/1%SDS room temp 2x20min.
0.1xSSC/1%SDS 55C for 30 min.
the signal was a blowout within 5 hours.
general questions: did I use too much probe?
were wash conditions addequate?
any other helpful hints appreciated.
respond here or with email
thanks in advance
the slackful Jamaican
Dept. Cell Biology
Baylor College of Medicine
mg690769 at mbcr.bcm.tmc.edu
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