Hi there, and thanks in advance for any help!
For several months, I've been trying to do some basic cloning of a 300bp insert from BL21 genomic DNA into a pET28 vector.
The PCR of the insert using Phusion Flex Hot Start seems to give a product, which I purify either with a kit or with electrophoresis.
I do double digest of the vector and insert. A gel shows the vector is cut, and previous troubleshooting has shown that the individual restriction enzymes SacI and HindIII are both working.
I ligate using NEB T4 DNA ligase using a vector:insert molar ratio of between 3:1 and 1:3, and vector amount being 50ng in 20uL reaction.
I usually obtain colonies, and after mini prep and PCR screen with Phusion or Taq, get a positive result.
However, sequencing only ever shows uncut plasmid. What's going on?!
My only clue is that I'm using pET28a, but sequencing seems to show pET28b; ie, there's a frame shift between the His tags.
Thanks! I hope someone can help.