Overlap Extension / Fusion PCR - HELP

[bengen]

Hey Everybody

First i want to say hello, because its my first post here. Currently i am doing my master thesis, and in this context i am going crazy on a method called Overlap Extension PCR / Fusion - PCR / Splicing - PCR..

I have to use this method to create hybrid products, which would take ages to clone in the normal way. I already joined two fragments, with the size of 400bp and 2000bp, it was no problem. The 3' Primer of the first fragment, was overlapping (20bp) with the 5' Primer of the second frament, normal PCR with NEB Phusion Polymerase - 40 Cycles, Primer addition after the first 15 cycles. At the end i got a product at the first try, with a lot of yield.

But since several weeks i am trying to use this method with 2 Fragments, with almost the same size. One is 1,1kb, the other is 1,3kb. And i didn't get any product at all. I tried a thousand protocols, with Phusion Polymerase, Taq Polymerase, Pfu Polymerase. With and without Primers, much and less template... i am not getting the cause of the problem.
I amplify the template fragments with Phusion Polymerase, than gel extraction in case of many fragments caused by mis-priming or just normal purification with a Quiagen-Kit if there is only one fragment present in gel-electrophoresis.

Can somebody please help me, i am going mad at this. If you need more informations, just ask, you will get it.

Many thanks,
Ben

P.S.: Found this post about10540.html, but nothin helped yet...

[roxanalibe]

OVERLAP EXTENTION PCR

Date:
Mutant wanted:
Primers used:
PCR 1 (to obtain fragment 1 and fragment 2) - that you already have


5 μl Fwd (20 μM)
5 μl primer REV (20 μM)
2,5 μl dNTP
0,5 μl template DNA (500 ng)
10 μl DMSO
10 μl Polymerase Buffer (Mg)
1 μl Polymerase High Fidelity
66 μl water


CYCLING
94 °C 2 min

95 °C 45 sec
57 °C 45 sec - Temp is depending on your melting temp of the primers
72 °C 2 min (1 min/kb PCR target)

72 °C 10 min
4 °C PAUSE


GEL purification:
- 1% agarose gel with big well for 100 μl – use 10 μl loading gel, 10 μl Smart Marker
- migration 20-30 min, 100V => take the band of interest
- purifier l’ADN using the protocole of QUIAGEN PURIFICATION KIT _- elution in 50 ul

PCR 2 - Fragment 1 +2

5 μl Fwd (20 μM)
5 μl Revers (20 μM)
5 μl fragment 1 DNA compare the size and the conc – molec to molec
5 μl fragment 2 DNA
2,5 - 3 μl dNTP - you could use the dNTP mix from Quiagen Mutagenesis kit (they are very good)
10 μl DMSO
10 μl Polymerase Buffer (Mg)
1 μl Polymerase High Fidelity
56,5 μl water



CYCLING
94 °C 2 min

95 °C 45 sec
57-62 °C 45 sec 25 X - USE A HIGH temp (65,70°C) if the size of the fragments is aprox the same
72 °C 2 min (1 min/kb PCR target)

72 °C 10 min
4 °C PAUSE

GEL purification:
- 1% agarose gel with big well for 100 μl – use 10 μl loading gel, 10 μl Smart Marker
- migration 20-30 min, 100V => take the band of interst
- purifier l’ADN using the protocole of QUIAGEN PURIFICATION KIT – elution in 50 ul




I had the same problem, with the overlapping of 2 fragment that where very close in size, it worked only when I performed the PCR with a temp of annealing higher than the one used to obtain each fragment.



Good luck,

Roxana