i am trying to fuse two gene, a GFP gene with another gene, lets designated the second gene as X.
i constructed a three primers for this purpose. 2 primers, will flank one end of GFP, the other primer will flank the other end of gene X.While, the third gene will be 'a bridge' between this two gene.
Commonly, to fuse gene, we constructed four primers, but i would like to try a three primer method, as i think it will give the same result.
The orientation of these fusion genes will be,
PRIMER 1-----X gene---PRIMER 2---GFP------PRIMER 3
Right now, i have got,
PRIMER 1-----X gene----PRIMER 2 at annealing temperature 50c. i got a clean, bright band without any smear or other bands.
but i am having problem to fuse it with GFP gene. The Tm of all the primers as followed,
PRIMER 1 71.7c
PRIMER 2 67.6c
PRIMER 3 64.6c
i have try different annealing temperature, from 60c to 50c
i have try a new template, new primers
it always end up with a blank gel (no band, smear or what so ever)
and finally i try a touchdown pcr, with annealing temperature ranging from 60c to 45c, with 1c/cycle.
for this TD pcr, i made two tubes,
one with all 3 primers and all templates (GFP and Xgene)
the second tube, contain PRIMER1----X gene----PRIMER 2 and GFP with PRIMER 3
i got a multiple band for tube number one, with of the bands is at the right size (~1.2Kb), so i going to purified it and run a pcr again.
but for the second tube, i got only the X gene!!!!, so i am confuse, as if the X gene is there, shouldn't the GFP gene is there also?
so i checked the primers sequence again, and i rechecked with colleagues and supervisor. The primers were correct
i didnt purify the result i got from the 1st tube yet, but i would like to know any suggestion or experiences from anyone who have this sort of problem before.
if i still didnt get this fusion gene, i think better of order a new set of primer with RE and then just ligate them together.
so please happy to criticize me if i am wrong!
Edited by thefallengrace, 04 July 2011 - 12:59 AM.