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I have been having trouble with what should be a fairly simple ligation. I had a plasmid that I needed to cut part out of, so I designed a couple PCR primers to do that. The plasmid already had a BamHI site on 1 side of the region I wanted to remove, so I made the other PCR primer with a BamHI site on its 5' end with 3 extra Cs so the restriction enzyme would bind better. Here is a crude diagram of what I tried to do:

enter image description here

So I did the PCR and BamHI digest, it looked good on gel, so I purified it from the gel, got the concentration on the Nanodrop, and set up a ligation.

However, I keep getting zero colonies. I have tried a 24 hour reaction at 4 degrees, and 10 minute reaction at room temp. I am currently trying a reaction where I supplement the buffer with fresh ATP, because the buffers are a little old. If that works, problem solved, if not, I am asking how to troubleshoot this.

This seems like it should be relatively simple, because I have no insert, just trying to close an empty vector, which seems to happen on its own when I don't want it to and dephosphorylate the vector.

UPDATE:

The reaction with extra ATP did not produce colonies. I will keep trying. One idea I had was to do the PCR and then try blunt end ligation, since the Pfu polymerase should make blunt ends. Also got a new sample of ligase.

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  • $\begingroup$ Can you heat inhibit your restriction enzyme and go straight to ligation without the purification step? I am asking because gel purification (or column purification) sometimes trim a bit the end and interfere with the ligation. This is quite rare but might have happen to you. $\endgroup$ – cagliari2005 Apr 9 '15 at 17:18
  • $\begingroup$ @cagliari2005 According the NEB poster on my wall, BamHI probably can't be heat denatured. $\endgroup$ – user137 Apr 9 '15 at 17:40
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    $\begingroup$ 1. How do you know the BamHI enzyme is working? 2. How do you know the ligase is working? 3. How do you know the cells are actually competent? 4. What is the selectable marker--is that the same drug in your plates? $\endgroup$ – mdperry Apr 10 '15 at 1:44
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    $\begingroup$ Hmm. According to NEB this should be ok. $\endgroup$ – Chris Apr 14 '15 at 16:58
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    $\begingroup$ @MarchHo After thinking about it a little more and writing the sequences on strips of paper, cutting the ends and wrapping them in a circle, I'm not so sure I was wrong the first time. Either way, I already ordered the new primer, will give it a try. $\endgroup$ – user137 Apr 15 '15 at 17:24

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