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plasmamembrane

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  1. 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
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