Help - Troubleshooting cloning into pET28 - all screens positive, but sequencing - (Oct/22/2012 )

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.

Your PCR screen of your miniprep may be giving false positives. You should do a negative control for your PCR. Do the primers bind once in the insert, and a second time on the vector? The PCR is done on the miniprep'd DNA, correct?
Have you digested the miniprep DNA to see if it has bands the correct length? Do the same cuts with pET28a and run both on a gel.

Perhaps your PCR product is not what you expect (maybe part of pET28a?)

Do you have 5' overhangs before your restriction site in your PCR primers for the insert? It's hard to tell if a PCR product is being cut.

Use NEB cutter for the Vector + Insert sequence. Next, find an enzyme that will produce 2-3 bands that can easily be distinguished (i.e. 700bp, 2000bp, 5000bp). Do the restriction digest and run it on a gel and this will tell you if you have the insert or not. Then you can sequence it to make sure the orientation is correct. PCR can give strange results when you are screening colonies and that is why I only use colony PCR as a preliminary screening technique and then use restriction digests to confirm before sequencing.

Also, the frameshift would only become problematic for expression.