3'end mapping

lr50916 at glaxowellcome.com lr50916 at glaxowellcome.com
Thu Jan 29 08:08:02 EST 1998


patcharaporn deshnium wrote:
> 
> Dear Friends,
> 
> I 'd like to consult you about the method of 3'end mapping. I tried to map
> 3'end of mRNA by S1 nuclease using double stranded DNA as probe. But, I
> didn't get a good result from this method. If you have a better way to map
> 3' end of mRNA, please forward to me. Thank you very much.
> 
> With my best regards,
> 
> Patcharaporn Deshnium
> 
> King Mongkut's Institute of Technology Thonburi
> Bangmod, Rasburana 10140
> Bangkok, Thailand

Well, I have found S1 nuclease assay with a 3' end labeled probe most
valuable to map the 3' end of genes. You should try and figure out why
your experiment did not work. 
1. quality of probe - test run a sample on an acrylamide gel
2. hybridization conditions - hybridize overnight RNA with probe at
various conditions, I did my RNA hybs at 56C because of the high GC
content.
3. the quality of the hyb solution, make fresh solution, aliquot and
store at -20C
4. quality of RNA

S1 nuclease is the best assay to map the 3' ends of RNAs. Don't give up.
What RNA are you trying to map the 3' end of?

good luck!

LR



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