primer design problem - (Aug/18/2005 )
i am designing primers using a 1500bp gene into the pET15b vector. i would like to use the NcoI cut site as the 5' primer, but the cut site is CCATGG (cutting between the CC and GG). i am worried that the ATG in the middle will act as the start codon and the protein will be out of frame when transcribed.
the first couple of codons are: ATG ACT TCC GCT GAG
i thought about using the G (in the GG part of CCATGG) as the first base in the next codon making it GTG or even omitting the ATG and making it GCT, but these codons translate into the wrong amino acids. is there any alternatives?
I assume that you are trying to ligate into a vector with an NcoI site at the appropriate location??
If this is the case, another alternative is to use a completely different site for your primer-a blunt end cutter- and chew back the ncoI site in the vector (should be a 3' overhang if I am understanding your question) This leaves only one C from the NcoI site and you could blunt end your product and ligate to that side.
You would still have compatible sticky ends on the 3' end of your gene so it won't be a completely blunt-end reaction and would still give a better chance for cloning in the correct orientation....
hope that helps, good luck!