Poblem with extended PCR

[k.d.preuss]

Hi netters,

I have a problem making extended PCR. I am able to produce two PCR 
fragments each by using specific primers. Both fragments (each around 1 kb)
have an overlapping region of about 80 nt and I want to fuse them by PCR.

     P1->
f1:  ======================================
                                       <-P2
                                 P3->
                          f2:    =================================     
                                                              <-P4

I made asymetric PCR using f1 with P1 and f2 with P4. After purifying
the products by agarose gels I mixed f1asym and f2asym. After denaturation 
and  reannealing I made 10 cycles 94!C 1 min, 72!C 2 min. Then I added
P1 and P4 and made 30 cycles (94!C 30sec, 55!C 30 sec, 72!C 2 min).
No full length product is detectable even by hybridization.    

Questions:
- Why isn't it working?
- Does anybody have a real working protocol?


Please answer to tm12kpkp at rz.uni-sb.de

Thanks in advance
Dieter


 
 
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