Cloning two fragments into a vector - (Oct/21/2011 )

Hello all,

A general question - To clone two fragments A and B (contained in two different vectors V1 and V2) into a vector (V3) which method would be the best?

1. PCR amplify the fragments of interest from each vector in which it is present by adding restriction sites of interest at the ends (with those extra base pairs); digest both fragments A and B with enzymes, ligate into a single fragment (A + B ) and then clone it into the V3. or

2. PCR amplify A and B from the vectors V1 and V2 by insertion of restriction sites at 5' end of A and 3' prime end of B; overlap PCR using overlaping primers, PCR amplify A+B after overlap PCR; restriction digest the A+B fragment (and vector also) and clone it into V3?

3. prepare vector, A, and B as above and do a triple ligation reaction. If you have good vector and A and B DNA samples and properly cut ends, then triple ligation is quite easy. I'd recommend cloning into a vector with different antibiotic resistance than the ones containing A and B. You can reduce vector only background by using PCR to amplify your vector as well, and then digesting with DpnI to remove undigested template DNA. If you do this with enzymes that can be heat killed, you don't even need to purify your digestion products.

Thank you Phage434! Have never tried triple ligation before but want to try it next time. One thing is not very clear, since A nd B are removed from vectors V1 and V2, why it is better to use a different antibiotic resistance vector for cloning?

A different antibiotic eliminates background from undigested or religated vector of parts A and B.