ligation problems

tracy a davis davist at rockvax.rockefeller.edu
Sat Sep 23 18:15:05 EST 1995


hi ligators,
some points re blunt end ligs:
-blunt is 100x less efficient than sticky, no matter what u do.
-buy 10x T4 ligasr from NEB
-use 10x more insert over vector
-increase your DNA-conc by factor 10 altogether
-don't use PEG together with T4lig!
-make absolutly sure that your cut vec gives no colonies in a control
 reaction without insert AND ligase!
 thats cuz transformationeff. is much higher than bluntlig-eff.
-heat the vec to 65 for 5 min before adding to lig
-follow recomms about repurification, BSA inclusion, new DTT when still
  no colonies
-good luck





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