fusion pcr - (Apr/07/2004 )
I have the following problem:
outgoing from 2 receptors i want to make a chimera with the Extracellular/Juxtamembrane/Transmembrane Part from receptor A (ca. 2.5kb) and the Kinase-Part from Receptor B (ca. 1kb).
Does anybody knows weather i can do this by using fusion pcr and where i could get a protocol for that?
It wont work via inserting restriction enzyme sites, since i would have bad mutations in the fusing area and it would use too much time...
is there a more "elegant" way to do this?
thanks in advance!
Check this http://www.biols.susx.ac.uk/gdsc/tony/meth...thod_fusion.htm
To obtain the constructs for integrative transformation, we use fusion PCR. Briefly, three separate PCR reactions were performed. One PCR reaction produced the tagging construct, using primers tag-F and tag-R (see table 1). A second PCR reaction, using primers up-F and up-R specific for the gene, produced a 500bp region of homology upstream of the ATG of the gene to be tagged and included a 24bp overhang with homology to the beginning of the tagging construct. A third PCR reaction, using primers do-F and do-R specific for the gene, produced the ATG plus a 500bp region of homology within the gene to be tagged and also included a 24bp overhang with homology to the end of the tagging construct (see table 1). To fuse these three PCR products, they are gel purified and fusion PCR was performed as follows:
100ng of DNA of each of the three PCR products was mixed with 5µl standard Pwo polymerase buffer containing MgSO4 and 0.2mM of each dNTP in 0.5 ml PCR tubes (Greiner).
dH2O was added to a final volume of 49µl and the PCR mixture was heated to 94°C before 1µl Pwo polymerase (Roche) was added.
5 cycles of 94°C - 30 sec, 55°C - 1 min and 72°C - 3.5 min were executed.
Following these 5 cycles, the reactions were heated to 94°C and a mixture containing 5µl standard Pwo polymerase buffer including MgSO4, 0.2mM of each dNTP, 0.5 µM each of the most 5? and 3? terminal primer, 1µl Pwo polymerase in dH2O
(final volume of 50µl) was added. 25 cycles of 94°C - 30 sec, 55°C - 1 min and 72°C - 3.5 min were executed.
This resulted in a PCR product containing 500bp upstream of the ATG of the gene to be tagged, the tagging construct and the first 5? 500bp of the ORF to be tagged.