- ligase supposedly inhibits transformation of dna, so it should always be heat-inactivated at 65 C for 10 min
- NEB sells T4 DNA ligase at two concentrations: 400,000 units/ml and 2,000,000 units/ml. Jen Braff was using the 2,000,000 units/ml ligase and found that a 1:5 dilution resulted in about 30x more colonies. Also mentions phenol extraction/precipitation to improve transformation efficiency, from JGI library protocols (?). (all in email to Jason)
- NEB offers a Quick Ligation Kit, with a buffer containing PEG. PEG when heat-inactivated inhibits transformation, so the ligation has to be purified instead. Endy Lab has the normal ligation kit without PEG, so shouldn't have to worry about this.
- comparison of undiluted vs. 1:10 dilution of ligase (using 400,000 units/ml ligase stock) for one of my libraries showed about 3.5x more colonies for the undiluted ligase (see my notes for August 23)
- amount of DNA
- NEB recommends between 1-10 ug/ml of DNA in a ligation reaction, so originally I was trying to shoot for somewhere in the middle. I wasn't getting very many colonies (a few at most), so I tried digesting more DNA and ligating higher concentrations, which improved things a little. Found that DNA recovery efficiencies from gel extractions were much lower than expected.
- Ended up just PCR cleaning the digested backbone (P1010 insert) and the digested PCR'ed insert and using as much as possible in a 20-ul ligation reaction (~2.8 ug backbone + ~0.85 ug insert, way over maximum NEB recommendation) and ended up with libraries between 1e3 and 4e4, which was comparable to what it was using gel-extracted backbone