11 November 2015 2 7K Report

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!  

More Levi Adams's questions See All
Similar questions and discussions