1 reply to this topic

[morrison]

Hi

I´m doing 5´Race (kit from BD Biosciences) and i got a problem with specificity. My target is expressed at low level. After nested PCR I get several bands but all of them aren´t the wanted ones. The cDNA is fine. I tested it with a gene specific primer pair and got the right product. The control Race experiment also works fine. But if I use only one GSP (in the Race) the specificity is not sufficient any more. I think that somehow the specificity of the nested or the Race PCR could be enhanced.

So, can anyone give me a hint how to get my product?

Here my current PCR conditions:

Primers: 50 pmol/reaction

2 min  94°C

35 times:
30 sec 94 °C
30 sec 62 °C
1.5 min 72°C

10 min 72°C

Many thanks

Thilo

[caro]

Hello

I also use BD smart RACE, but your annealing temp are much too low. I would order new primers with Tm > 70 degrees, around 72 degrees and do a touch down PCR, and perhaps in both the first PCR reaction and the nested PCR.

Good luck (you may need it, 5' RACE is a pain in the a..)

Regards Caro