I have my protein of interest in pcDNA3 with Flag in the N terminus, and I want to create an untagged version of my protein. I designed some primers to amplify just my sequence,adding some restriction sites and introduce it in pcDNA3.
In order to do so, I added 5 bases (to allow the enzyme to cut), an EcoRI site and a kozak sequence in the Sense primer ( 17 extra bases, and 41 matched bases, 52% CG in total) and a HindIII site and 5 more bases in the Antisese oligo (11 extra bases and 43 matched bases,53% CG in total). Here my primers:
SENSE: 5´- GCAGAGCGAATTCCACCATGGCCTCACATGTGCAAGTTTTCTCCCCTCACACCCTTCAATC -3´
ANTISENSE: 5´- CCCTCAAGCTTTTATATGTAAGGGTACTGGTTGACCTTGGCGGGGCTCAGTGGG -3´
The Tm without the extra bases are about 84ºC
My protein is about 3600bp. My questions is:
Are those primers good enough to perform the pcr, and if so, how should I perform it? I was gonna do something like this:
Repeat steps 2-4 seven times
72 10 min
Is this the best way to perform that kind of PCR??
Thanks a lot
Edited by laurequillo, 27 October 2009 - 02:00 AM.