5' RACE......2* structure? - (May/02/2006 )

Hi,

I've read some of the responses dealing with 5' RACE, but didn't see anything about possible secondary structure. I've been having problems getting anything on my gels for 5' RACE (even using touchdown pcr). I think that it may be due to 2* structure. Does anyone have any protocols for relaxing the 2*?

Or.....What are some other tricks about the 5' end that would make my head hurt?

thanks,

matt

-grahamm-

Hi, I am doing the 5' RACE now. It is very important to do nested PCR.

Good luck!

Yin

QUOTE (grahamm @ May 2 2006, 02:38 PM)

-yin-

QUOTE (grahamm @ May 2 2006, 11:38 PM)

I don't have a lot of experience with 5'-RACE (in fact it's not working well for my gene), but to avoid trouble derived from secondary structure it should be good to perform the RT at high temperatures (up to 55ÂºC) and maybe use a PCR kit for GC-rich templates.

-drfelip-

I had a similar problem years ago doing transcript mapping. What I used was a low temp thermocycling approach to get through the secondary structure. I can't remember the exact details of the cycling conditions but they were described in the following paper

Optimized rapid amplification of cDNA ends (RACE) for mapping bacterial mRNA transcripts

Daniel

-Daniel Tillett-

QUOTE (Daniel Tillett @ Jun 19 2006, 02:52 PM)

It seems that this paper is not available online. Is there any other way of picking it up?

-drfelip-

QUOTE (drfelip @ Dec 13 2006, 04:51 AM)

QUOTE (Daniel Tillett @ Jun 19 2006, 02:52 PM)

It seems that this paper is not available online. Is there any other way of picking it up?

PM and I will send you a copy.

Cheers

Daniel

-Daniel Tillett-