How good is your cloning efficiency? - (May/13/2015 )

Hi, in my lab I do lot of cloning and sometimes the cloning doesn't work, low vector yield after digestion, pcr problems... I'm wondering how the things are working in your lab. Thanks!

Depends on what you're cloning but efficiency should be pretty high as long as your kit is high quality. The worst efficiency I get is 1 in 20 positive colonies on a plate. If I'm getting anything lower I tend to start again and design new primers for PCR prior to cloning. Are you using gene specific primers or vector-specific primers? Also are you using some sort of screening such as X-gal?    

It depends on the fragments. I have had 100% ligation efficiency from testing 48 colonies by colony PCR. I also have had ligation efficiency of 1 in 72 colonies tested.

It very much depends on the size of your fragments, the quality of your DNA, the quality of your ligase, ligase buffer and I have the suspicions the secondary structure that the DNA fragment adopts.