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Extra longer unspecific product of PCR - PCR (Jun/06/2007 )

Hi, I try to amplify the RANKL for cloning. But there is something strange. I get my product and get a longer unspecific band either---4000bp with the annealing tem 55, but the target gene is only 2400bp. what's the problem? I add extra bp at primer 5' for restriction enzyme site. and I didn't use touch down PCR. At the same time, I caculate my primer annealing time, It is should be 60.8 for the first several cycles and then 71 for the last cycles ( because there is extra base on the primer 5'). If i am going to try touch down PCR, it should be 60.8--71, 55-65 or 55--71? Is it helpful to get rid of the longer unspecific product?

-H.RUG-

What are you using as your template? Could you be using too much of a ~4kB plasmid? Also, how long do you spend in the elongation step? 2-2:30 minutes should be plenty, and might help minimize the 4kB band if it is just nonspecific.


Amanda


QUOTE (H.RUG @ Jun 6 2007, 01:14 PM)
Hi, I try to amplify the RANKL for cloning. But there is something strange. I get my product and get a longer unspecific band either---4000bp with the annealing tem 55, but the target gene is only 2400bp. what's the problem? I add extra bp at primer 5' for restriction enzyme site. and I didn't use touch down PCR. At the same time, I caculate my primer annealing time, It is should be 60.8 for the first several cycles and then 71 for the last cycles ( because there is extra base on the primer 5'). If i am going to try touch down PCR, it should be 60.8--71, 55-65 or 55--71? Is it helpful to get rid of the longer unspecific product?

-Cheamps-