riboprobes for northern blots

Michael Gerdes mg690769 at bcm.tmc.edu
Tue May 23 09:18:35 EST 1995


Help!
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

			Michael Gerdes
			the slackful Jamaican
			Dept. Cell Biology
			Baylor College of Medicine
			Houston, TX

			mg690769 at mbcr.bcm.tmc.edu




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