The "RACE" for 5'ends

[Matthias Zeiner]

Hi, 

I'm currently cloning a protein, but the cDNA still lacks it's 5'end.
Northern blots showed me that I am quite close (about 100 bp).

So I tried RACE, using the " 5'amplifinder" kit from Clontech (it uses a 
modified "SLIC" protocol).
When I followed the instruction manual it didn't work at all. So I 
changed the protocol and did 2 subsequent PCR's using 2 nested primers 
instead of 1. This gave me a beautiful band of the expected size on the 
agarose gel but sequencing of the fragment revealed, that it is absolutely 
unspecific (the primers on both ends of the PCR - product are ok).

Controls using internal primers show, that the reverse transcription step 
worked and the cDNA should be good. It seems to me that the linker 
ligation is not efficient.

Did anybody have success with Clontech's amplifinder kit?
I know there is a similar kit from BRL, is that one ok?
How could I improve the "RACE" technology?

Any suggestions will be greatly appreciated!

Matthias Zeiner
Inst Biol Chem
INF 501
D-69120 Heidelberg

mzei at sun0.urz.uni-heidelberg.de