Clontech PCR-Select subtractive Kit

Marilyn Bakker Marilyn.Bakker at med.monash.edu.au
Thu Jan 23 14:30:31 EST 1997


Dr. B. Popping wrote:
> 
> Dear Netters,
> 
> last week I posted an article asking about your experience with the
> Clontech PCR-Select kit (suppresser subtractive hybridization).
> The only replies I had so far were people asking to forward any
> information I'd get to them.
> 
> I can't quite believe that no-one has used the kit.  Or is the experience
> you have made with the kit so BAD that you do not want to share it ?
> 
> I'm still hoping to get information from poeple who have used the kit,
> even if they made had a negative experience.
> 
> Regards,
> 
> Bert Popping
> 
> --
> Bert Popping
> E-MAIL: bpopping at hgmp.mrc.ac.uk
Hi Bert.
I am currently trying to use the Clontech PCR Select kit.
So far no luck, and I think you need a lot of that.
I have been failing at the adapter ligation step. Control works, but
experimental doesn't. Instructions say "repeat ligation" ???? Repeat
something that hasn't worked without changing anything????? I don't
think so. So I increased the amount of tester in the ligation. Worse
result!
Increase the amount of ligase - worse result. G3PDH control primers work
on both samples, but I am surprised that the standard control in such a
kit amplifies exactly the same size from CDNA as from DNA. Not a good
control in my book.
The PCR primers have worked slightly in the 1st tester control ligation
(both primers) but only primer 2 in subsequent ligation  reactions.Maybe
there has been too much ligation, so all I'm getting is total
suppression?
Anyway its back to square 1 to obtain more poly A and start again. 
Trouble is, this kit is extraordinarily expensive in Australia ($1300)
and they give you  a very mingy amount of ingredients that allows no
room for failure.
If I were  starting again I would probably use the original Hubank and
Schatz method. N.A.R. 1994,vol22, no. 25 pp 5640-5648.
I likewise would be interested to hear other peoples experiences.



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