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Fusion PCR Protocol? - (Sep/22/2003 )

I am looking for a PCR Fusion protocol. (using pcr to make a fusion protein) Does anyone know where I can find one?
Any help would be appreciated. Thanks

-bouttela-

QUOTE (bouttela @ Sep 22 2003, 04:21 AM)
I am looking for a PCR Fusion protocol. (using pcr to make a fusion protein) Does anyone know where I can find one?
Any help would be appreciated.  Thanks

I am trying to do the same. It worked for me with short sequences, but now is a headake. If you find something better, please, tell me!:
Splicing by overlap extention by PCR.......
A Warrens, M Jones and R. Lechler
Gene 186 (1997) 29-35
and references therin

-ale-

I have done this many times and without much problem. Don't really have a protocol though.
What i do is this:
make one forward primer(A) for the 5' fragment. make one reverse primer(cool.gif for the 3' fragment. So far its easy. Then, make a reverse primer© of the 5' fragment with an extension complimentary to the 3' fragment. Make a forward primer(D) for the 3' fragment with an extention complimentary to the 5' frament. (so if you design your primers from 5' to 3' the complimentary region is on the 5' end of both primers)

Then you PCR the 5' fragment with primers A and C and in a seperate tube PCR the 3' fragment using primers B and D. You do not need to get much of these fragments, so don't over PCR you'll just introduce mutations.

Load the samples on a clean gel And carefully cut out the two bands (LMP gel is best for this but a normal gel works fine also). Just cut out the most concentrated part in the gel no extra agarose. Add ~ 10X volume water, melt the agarose,mix and add ~3ul of each fragment solution to a new PCR mix (100ul total) (containing primes A and B only). Mix and place in PCR machine. heat to 94C 1', then cycle 94C 1' anneal 3' at atleast 5C below the overlap homology mt temperature extend longer then normal repeat 4X. Then PCR with correct conditions for primers A and B.

The gel part is importent to lose the primers and the original template.

-il0postino-

Disruption by Fusion PCR
David Amberg and Ellen Beasley
1) In separate PCR reactions, amplify the 5' and 3' ends of the gene of interest with primers about 200 bases apart. Primer 2 should begin with 24 nts complementary to the m13 forward primer(GTC GTG ACT GGG AAA ACC CTG GCG) and primer 3 should begin with 24 nts complementary to the m13 reverse primer (TCC TGT GTG AAA TTG TTA TCC GCT). PCR amplify the marker using the m13 forward and reverse primers. The Prakash and Jones vectors are useful for the marker PCR. The conditions for these PCR reactions are as follows:



20ng Plasmid template DNA
5ul 5uM Primer1/ m13 forward/ Primer3
5ul 5um Primer2/ m13 reverse/ Primer 4
5ul 10xVent Buffer
5ul 2mM dNTPs
1ul Vent
H2O to 50ul
10X Vent Buffer:
0.2M TRIS pH 8.8
0.1M KCl
0.1M (NH4)2SO4
0.02M MgSO4
1% Triton X-100
Do 94Cx4 min. then 30 cycles of: (94Cx1min. then 55Cx1min. then 72C for 3min) finish with 72Cx20min and a 4C soak.

2) Gel purify all the PCR fragments on low melt agarose.

3) Set up the first fusion PCR by melting the 5' end fragment and marker fragment at 65C and adding to a PCR reaction as follows:



2.5ul of each fragment
5ul 5uM Primer1
5ul 5uM m13 reverse primer
5ul 10xTaq Buffer
5ul 2mM dNTPs
1ul Taq
H2O to 50ul
10X TAQ Buffer:
0.2M TRIS pH8.3
15mM MgCl2
0.25M KCl
0.5% Tween20
Do 94Cx4 min. then 30 cycles of: (94Cx1min. then 55Cx1min. then 72C for 3min) finish with 72Cx20min and a 4C soak.

4) Gel purify the product of the first fusion PCR on low melt agarose.

5) Set up the second fusion PCR by melting the product of the first fusion PCR and the 3' end fragments at 65C and adding to a PCR reaction as follows:



5ul of each fragment
10ul 5uM Primer1
10ul 5um Primer4
10ul 10xTaq Buffer
10ul 2mM dNTPs
2ul Taq
H2O to 100ul
Do 94Cx4 min. then 30 cycles of: (94Cx1min. then 55Cx1min. then 72C for 3min) finish with 72Cx20min and a 4C soak.

6) Check for product on a mini gel. Phenol/chloroform extract the product and EtOH pptate and use directly to transform a diploid or gel purify the product and transform the diploid with this DNA.

Note: If PCR generated errors are of concern then each PCR generated fragment can be extracted from the low melt agarose and EtOH precipitated. Once free of the agarose, Vent should work in the fusion PCR reactions.

Take a look at this please, good luck!

-nostaljikkedi-