Poblem with extended PCR

k.d.preuss tm12kpkp at rz.uni-sb.de
Thu Feb 2 10:46:24 EST 1995

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.

f1:  ======================================
                          f2:    =================================     

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.    

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

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

Thanks in advance

*  Dr.K.D.Preuss                   E-MAIL: tm12kpkp at rz.uni-sb.de *
*  Physiologie II                  Phone: +6841-166123           *
*  Universitaet d. Saarlandes      FAX: +6841-166655             *
*  66421 Homburg/Saar                                            *
*  Germany                                                       *

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