5' RACE Specificity Problems

[Shahram Mori]

Sarah Childs (childs at oci.utoronto.ca) wrote:
: I'm having trouble with specificity in 5'RACE.  

: I'm getting non-specific bands, even though my gene-specific 
: primer is an 18 mer with a Tm of 58C, I'm doing the PCR with a hot start and 
: annealing at 56C.  At the same time I am not getting the product I want.  
: Has anyone had similar problems and overcome them?  Does anyone have a 
: very good RACE protocol that might help me?

: Sarah Childs
: Ontario Cancer Institute

: email: childs at oci.utoronto.ca
The problem is probably at your anchor ligation Sarah. I used Clontech's
5'RACE but I never got anything. For very few people it worked.I would
make sure that you are getting your anchor ligated to the 5' ends.
Cheers,
 --
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_ Saskatoon/SK/CANADA
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
88003