Ligase

1) first of all, as a random fact: the optimum temperature for t4 ligase is 16 centigrade (i don't know why, so don't ask me). Now, ALL PROTEINS have to be kept on ice AT ALL TIMES. if you leave them at higher temperature that that (even RT) they misfold ...

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Thank you so much for replying and laying out possibilities that could be helpful. 1) Not that I left my ligase on the desk unattended but about your point 1-" if the ligase has been left on the bench for even a couple of minutes, it's pretty much lost." My question ...

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... you seem to use very low ligation ratios. I usually do a 16:1 ratio to get as many colonies as possible. What you could try: 1. Throw away your T4 ligase and buffer and get new ones. this probably is not the problem, but if the ligase has been left on the bench for even a couple of minutes, it's ...

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... transformations using the DH5alpha high transformation efficiency cells (NEB). None of these ratios worked- no colonies. I had used the same T4 ligase enzyme and buffer that worked fine couple weeks before. I made sure that the buffer had no precipitate and mixed it well. I also tried a different ...

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... VαCα cassette with 5 codon degeneracy spanning CDR3α was ligated into the MSCV vector as a AgeI-NotI fragment. Ligation was performed with T4 DNA ligase (New England Biolabs) with a 2/1 insert to vector ratio.

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