In article <9308101839.AA01473 at cardinal.ncsc.org>,
mbwhite at CARDINAL.NCSC.ORG (Martyn White) wrote:
> So I have two questions
> 1) Those of you who have succeeded in getting your 5' end.
> What do you think is the best approach?
>> and secondly and less importantly
>> Do clontech suck?
Clontech do suck!
I tried getting the manual (recipe book) from them so that I could perform
the modified SLIC reaction myself. They were as tight as a duck's ass (and
that's water tight!)
We have performed SLIC in our laboratory. It is a very mystical reaction. I
have managed to get a single 5' product from one reaction and one reaction
only. This was not reproducible. Another person in the lab. swears by the
technique (and we swear at her).
My question is, why do you want the 5' end? If you need to map it, for
example, for transcriptional start site analysis, then why not try primer
extension, S1 nuclease or preferably not RNAse protection analysis. Do not
do the last one although it is an option.
The strategy that I have adopted to map the transcriptional start site is
to isolate sequence upstream of the 5' end, then to create various 3'
deletion mutants of the fragment until I have chopped off the
transcriptional start site - measured by transfection studies.
I will then go back to the multiple bands seen with the aforementioned
techniques and correlate them with the transfection studies.
If you can think of anything better please let me know.
Good luck banging your head against that wall.
E-mail: leach at mbcrr.harvard.edu
p.s. Clontech bloody overcharge on all their RNA's.