Hello,

I am doing cloning using pET28a vector, I repeated many times but couldn't get colony.

I digested the vector by BamHI and XhoI, and purified the gene, as well, digested the insert genes by these two RE enzymes at 37 c water bath for 3 hours. I checked after digestion, those digested very will.

I did the ligation and used 3 to 5 ratio over to plasmid vector. And inactivated at 65 c for 10 minutes.

The ligation time I did for overnight at 16 c, and at 22 c for 1to 3 hours.

I am using the BL21 as host.

I used 5 microliter for transformation, and had negative control as well. But I couldn't get any colony even for Negative control as well.

I did many times, still no colony

Please help me from your experiences

Thank you in advance