Posted 17 May 2009 - 07:40 AM
im trying to digest a vector and insert with ecori and hindiii enzyme and perform ligation.The Tm value for the EcoRI and HindIII cut sites is 16C .I have tried ligatin in lower temperature range but didnot suceed .Can I use the temperature between 16C and 25C to balance with the efficiency of ligase or it is not required ?Im confused with the temperature that is optimal for successful cloning.
Plz can anyone help??
Posted 17 May 2009 - 06:06 PM
I do all my ligations at 16C, I've never even calculated the Tm of a restriction site...
I guess what I'm saying is that if your cloning experiment failed, it is unlikely that the failure was due to your using a 16C ligation temperature.
Posted 18 May 2009 - 05:57 PM
Treat the vector with phosphatase to prevent recircularisation.