Long-distance PCR problem
Posted 12 October 2004 - 10:02 PM
the pcr programme is a TOUCH DOWN pcr. it60 is 94C 1mins and followed by 16 cycles of 98C 10sec 68C 9mins with 0.5C degree lower per cycle. ANd follewed by another 22 cycles of 98C 10sec , 60C 9mins. and 60C 10mins finally.
my primers are two GSPs and two adaptor primers. a nest pcr was done.
but the result was really disappointed. a smear appeared and the clear bands obtained were all small size.
would some guys give me some suggestions?
thanks a lot!
Posted 12 October 2004 - 10:35 PM
an 8`C drop in annealing temps sounds huge... are you sure that's right?
Posted 13 October 2004 - 02:12 AM
My cycles are always 30 sec melting (95 degrees)
30 sec annealing(varies with primer, touch down etc)
X min elongation (time varies with length and temp varies with the polymerase.
If you in each PCR uses a GSP and an adaptor it is really difficult to obtain anything, but higher anealing and more cycles might do the trick.
The touch down sound reasonable for programming reasons I often use two degrees per five cycles -less programming to do.
Posted 15 October 2004 - 12:49 AM
why an adaptor and GSP produce high anealing? isn't RACE based on this ?