It actually took me a day or so to accept my restriction digest had failed, and it took me a few hours to guess why it did not work. What I wanted to do was digest my plasmid and my gene with HindIII (in separate reaction tubes). This would leave HindIII sticky ends on both. At the same time that I was digesting the plasmid, I wanted to also dephosporylate it so it would not re-circularize (stick to itself during ligation). To verify that I cut the gene and the plasmid, I ran them on a 2% gel. I expected that if I ran to the end of the gel, I could get separation between the cut and uncut genes, as the difference is about 150 bp. I also expected the uncut pUC19 plasmid to run much faster than the cut pUC19. Alas, this was not the case.
What a mess. Seriously, this is a pretty gnarly gel. Anyway, from left to right the loaded lanes are:
- 2 log ladder
- un-cut bg fragment
- cut bg fragment 1
- cut bg fragment 2
- pUC (way overloaded…)
- cut pUC A
- cut pUCB
As you can see, the cut fragments of pUC ran faster than the uncut fragments. This is very suspect, since the supercoiled plasmid DNA should be “smaller” than the cut DNA. It was not a small amount either, it looks like it ran somewhere between 1k or 500 bp faster. This could be due to over loading but it is not a good sign.
The cut fragments of the gene did seem to run a little faster, but the gel is so gnarly it is hard to tell. They do look like maybe they got cut, but it is hard to tell when the bands are so misshapen and far apart. It would be no good to try to ligate them to an un-cut plasmid. So I need to troubleshoot this step.
What Is Wrong:
I strongly suspect that my restriction enzyme digest was foiled by the heated lid of the pcr machine. It heats by default to 114C or so, to prevent condensation on the lids of the tubes as you get up to 98C or so. I had it on at full blast- the inactivation temperature for HindIII is 80C, and I used the “time saver” protocol and only digested for 30 minutes. So what did not get inactivated had little time to get its enzymatic work done.