ligation of an adaptor into a vector - (Jul/25/2010 )
hello i have been trying to ligate an adaptor into a vector without success. first i treat the vector (puc 18) with sac1 enzyme (37 degrees for one hour or more for complete digestion), perform phenol chlorofoam extraction as well as ethanol precipitation. i then dephosphorylated with BAP (at 65 degrees) and again performed phenol chlorofoam extraction as well as ethanol precipitation. i also dephosphorylate by adaptor (sac1 sal1) with PNK before ligation. i then transform with DH5 alpha competent cells by chilling on ice for 30 mins, electroporating at 42 degrees for 2 mins and then chilling on ice for 1 minute. i then add 1ml soc and incubate at 37 degrees for 30 mins after which i plate onto agar plates containg ampicillin.
after several attempts my colonies do not contain the insert. i then tried ligating vector with insert with ligase and vector with insert without ligase. the number of colonies in the vector with insert without ligase was ten times more than the vector with insert with ligase.
can anyone help me with an explanation as to what is happening? thank you very much
I have been trying to ligate an adaptor into a vector without success. The DNA Ligation Kit is a simple two-component system that produces very rapid ligation reactions using T4 DNA Ligase and an optimized buffer system. Due to the high efficiency of the ligation reaction, conventional overnight incubations are no longer required.
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you do an electroporation for 2minutes?
Dont you mean heat shock?
I think Bobo ia talking about transformation by heat shock not electroporation.......
DNA, you are right. I was talking about transformation by heat shock and not electroporation. Sorry for the error
Thats what I was thinking too...
anyway: try an electroporation then.... Sometimes heat shock just wont work.