I have recently made a mutant library of a gene with DNA shuffling between two genes. There are 25 mutations in total and I tried to shuffle these two genes via StEP PCR. After restriction and ligation into vector, I got approximately 7000 clones, which I washed from plates and performed plasmid minipreps, pooled the minipreps in a total volume of approx 1000 ul (that way I have an amplified library stored as a plasmid in freezer).
Now I have to begin with screening, which I will do with activity assays in microtiter plates for ammonia release.
I transformed (chemically) 1 ul of my plamid library DNA, that gave me around 49,000 clones.
My Question is
How many clones shall I screen to give a good coverage of my library?
How many clones I should have got to have an idea about good shuffling after the first StEP PCR? I sequenced 20 clones and on average there are 3 crossovers on 1.5 kb long gene. in some cases, only one crossover and in some its 5 crossovers.
Screening a mutant amplified library
No replies to this topic