Hello,
I am trying to clone a 1.1Kb gene into 5.7Kb plasmid. It seems straight forward but I got no colony. Below is my protocol.
Insert preparation:
The insert was generated by PCR. RE sites ( BamH1 and HindIII ) were designed on 5' end of primer. I also generated extra 4 bases for efficient cutting.
The PCR was fine. I got a sharp band on gel. I cut the gel and purified it. The product was doubly digested ( BamH1-HF and HindIII-HF
from NEB in Cutsmart buffer for 15 min ). Then I gel purify it again.
Vector preparation:
Vector was also double-digested with two REs. I gel purify the DNA after digestion.
Ligation:
80ng of vector was used for ligation. Vector/insert ratio was 1:3.
ligation enzyme: NEB T4 DNA ligase
time: RT for 1hr then 65degC for 10min for inactivation
transformation:
DH5alpha ( competency: around 10^8 cfu/ug DNA )
2uL of ligation mixture was used for transformation
heat shock: 1min at 42 degC
recovery: 45min in SOC
incubation O/N at 37 degC on LB-antibiotic plate, but no growth.
Need your help.
Thanks a lot
