I am going to clone a gene into pET32 a expression vector. After completing the cloning process, I verified my insertion by PCR an I see about 800 bp product . Then also, I wanted to verify with restriction cut, but instead of two bands I got one band. I don't know where is the problem, can you help me
Serap Demirel Can you explain a bit further? What is the size of the band that you see on the gel? size of your plasmid minus insert?
hi
1- Your enzyme may not work ( you can used from Fast digest restriction enzyme from thermofisher as good enzymes )
2- check other restriction site on your vector
2-you can check gene direction verified used by s.tag primer -F & your insert-R ( if Tm primers close to each other ) or s.tag primer -F &T7-terminator-R
3- you can run colony-PCR in 25 cycle with Tm gradient program ( to get 1 band in PCR)
good luck
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