incorporation of restriction site in Primers - Help to design primer to clone gene in frame with fluorescent tag (Jul/07/2008 )
I need to make construct where my gene should should be in frame with mcherry, so that I can express the mCherry as well which is a red fluorophore.
Since Xho I site in vector is in triplets , i do not need to add any base to make up for making codon ,,,is this correct ???
Pls see attached file for vector sequence if needed, i need to incoporate 2 restriction sites sequence for cloning purpose so that my protein remains a N-terminal fusion to mcherry (vector)
i need Xho I at 5'end in fwd primer and EcoR I at 3' end of reverse primer
so i have designed primers like this..
Forward 5' XX Xho I sequence ATG..rest of my sequence (18 mer)
Reverse 5' EcoRI sequence end of my sequence (without stop codon 18 mer)
First, you need at least one additional base on your 5' to get efficient digestion with XhoI and that's only with a 20 hour digest (check out NEB catalog or website: cleavage close to the end of DNA fragments)!!! Next, if you are planning on trying to express this in cells you may want to include a Kozak sequence. The only other concern I would have is during transcription you may get two products, your insert fused to the tag and the tag alone. I've had this happen and had to go back and mutate the ATG on the tag.
Thanks rkay447, i see your point but wont high transfection efficieny will compensate for expression of insert fused with tag...
can u pls suggest me the primer sequence ,,,, if I want to use SmaI and Nco I in fwd and reverse primers respectively, my biggest concern is if Sma I is not in frame then the fusion protein will not be in frame. I am attaching the vector sequence for your reference.Thanks for your help!