Long-distance PCR problem - help (Oct/12/2004 )

i am now trying a long pcr. the product is estimated to be about 7K. and the polymerase i used is LA Taq from TAKARA.
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!

I though touchdown PCR involves decreasing the annealling temperature by 1 degree C every second cycle .... and then 'touchdown' annealing temp which is then used for 10 or so cycles.
an 8`C drop in annealing temps sounds huge... are you sure that's right?

vetticus

First check if your polymerase is a hot start, then you need 10 min 95 degrees to activate it (or you can leave it and make all your cycles at the same lowest temp, that is a bit like touvh down).
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.

Caro

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



why an adaptor and GSP produce high anealing? isn't RACE based on this ?