I'm trying to clone several genes into one plasmid, and was looking at using a 2A self cleaving sequence because its short. So:
start-Gene A -- 2A-- Gene B - stop
As far as the actual cloning - since the 2A sequence is around 60 bp, it seems economical to actually purchase 2 oligos corresponding to top and bottom and adapt a stitch-PCR technique to generate this construct in 2 steps with a high fidelity polymerase (like Q5) by designing overhangs (about 6-8 bp each) in the original oligos. i.e:
Gene A........ .....2AOligo2.......
.....2AOligo 1 ....... ....Gene B...
Does anyone have any particular experience doing this sort of thing or able to recommend a more effective design? I'm not sure if this will even work!
Clarification: I was thinking of combining each gene with its oligo in one stitch PCR, then trying to stitch PCR to two chimeric constructs together.
Hi Levi, I don't see any problem with your cloning strategy. Overlap of 6-8 nucleotides seems too short though. I always prefer to have an overlap of at least 10 nucleotides, ideally 15 nucleotides.
Hi,
I am about to do such a similar thing. I have a doubt in this.
My strategy is:
Promoter-GeneA-2A-GeneB-2A-GeneC-stop
Now I have designed overlap primers.
My doubt is my overlapping primers are only on the overhangs.
For example,
GeneA - Reverse overlapping primer
31bp of GeneA and 45bp of 2A sequence (in reverse)
GeneB - Forward overlapping primer:
Overhang (15 nucleotides preceding GeneA-60 nucleotides complete2A sequence)-
19 nucleotides of GeneB
So, for GeneB, there is an overhang of 75 nucleotides. This overhang overlaps with 15bp of GeneA and the 45bp of reverse 2A sequence.
This ultimately means that GeneB overlaps with the original GeneA only with 14 nucleotides. The rest of the overlap is only with the overhang of GeneB.
Will it be a problem?
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