I tried to clone a 1.1Kb gene into 5.7Kb vector but failed. Below is the protocol:
The insert was generated by PCR harboring BamH1 and HindIII restriction site. The primers were designed to have extra 4 bases for efficient RE cutting. I gel purified the DNA and set up restriction digestion with both enzymes ( BamH1-HF and HindIII-HF from NEB in Cutsmart buffer ). After digestion, I gel purified again.
The vector was prepared by digesting with both RE and purified by gel extraction. I saw a clear and sharp band on gel after RE but I did not check two REs individually.
80ng vector , insert/vector ratio: 3:1 ( evaluated by spectrophotometer at A260 )
ligase 1uL ( NEB, regular ligase )
reaction volume: 20uL
ligate at RT for 1hr, heat inactivate for 10min at 65 degC
DH5 alpha, competency around 10^7, prepared by Inonue method
heat shock: 42degC for 45 sec
recovery: SOC for 45min
2uL of ligation solution was used to transform E. coli
O/N at 37 degC on LB plate with selective antibiotics
No colony recovered!
Is it possible that UV exposure ruined the DNA?
Need your help. Thanks a lot.