Enhance specificity of 5īRace - (Jul/19/2004 )
Iīm doing 5īRace (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 arenīt 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
30 sec 94 °C
30 sec 62 °C
1.5 min 72°C
10 min 72°C
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..)