Wrong antibiotic

[jwb]

Hi!

Suppose I plated a ligation mixture on ampicillin instead of kanamycin, by error (which I've just managed to do!), and considering that I've heard ampicillin only inhibits growth but does not kill, is there any chance that replica plating might help me recover them from the amp plate on to a kan plate? or is replica plating only for grown colonies?

Dreading doing another round of ligation. I usually do not use the entire ligation mixture, but of all days, I did just that today!

[relaxin]

If you have plated out the transformed cell on ampicillin plate, you may recover the bacteria by flooding the plates with LB and scrape the surface of agar with a L-shape glass rod. Then transfer the bacterial suspension into a sterile tube and spin down the bacteria, wash once with LB and then plate on kanamycin plate. You may need to do this gently, as these transformed bacteria are quite weak.

[jwb]

Hi relaxin,
Thanks for replying. I'm doing that now. I'll post back on how it worked. Really, it is very difficult to motivate oneself to prepare for another ligation reaction with mistakes like this one!

[jwb]

This did not work.
I washed off the spread bacteria, centrifuged, and resuspended in some LB, but I did not wash twice out of fear of losing the scanty pellet.
Back to ligation again. Teaches me to keep spare ligation mix!

[talkingtree]

Perhaps you still have some cells left from incubation with SOC media after heat shock, those can be used.

[mchlbrmn]

If you use more than 5 or 10% ligation in a transformation, it's said to reduce the transformation efficiency (and reduce the colony # at some point).

[jwb]

Hi mchlbrmn,

I am the only person in the lab who keeps apart some ligation mix as back-up (which I regrettably didn't, this time).Everyone else uses the whole thing and they get enough colonies all the time.

One thing I feel though is that the ligation mixture has to be mixed once with the pipette before using it for transformation. On one occasion I remembered that my back-up mix(5 ul) gave me many more colonies (4-5 fold more) than my initial transformation mix (15 ul). This, I presumed, is because some DNA kept stationary for quite some time in the tube before transformation sinks down to the bottom. It sounds rubbish, really, but maybe it could happen. The competent cells I used were the same batch.

[relaxin]

Ligated DNA in solution is homogeneous. You really do not need to mix it before pipetting an aliquot for transformation. Unless you heat-kill the ligase (which will interfere with transformation), you need to mix the solution afer spinning down the condensed water at the cap of the tube.

With high transformation efficiency competent cells, you need only 10 ng of ligated vector DNA for each transformation. The remaining ligated DNA can be kept at -20 C until you can count the colonies.

[morkfromork]

Hi mchlbrmn,

I am the only person in the lab who keeps apart some ligation mix as back-up (which I regrettably didn't, this time).Everyone else uses the whole thing and they get enough colonies all the time.

One thing I feel though is that the ligation mixture has to be mixed once with the pipette before using it for transformation. On one occasion I remembered that my back-up mix(5 ul) gave me many more colonies (4-5 fold more) than my initial transformation mix (15 ul). This, I presumed, is because some DNA kept stationary for quite some time in the tube before transformation sinks down to the bottom. It sounds rubbish, really, but maybe it could happen. The competent cells I used were the same batch.

I've noticed that some ligases seem to require using alot of the ligation reaction to transform the bacteria. I always use fast-link ligase from Epicentre (CamBio in he UK), I have no connection to this company but this product works really well and it's relevant to your post so I thought I would recommend it. You can do transformations with 0.5ul of your 20ul reaction (I generally use 1ul). I usually use about 10ng of acceptor plasmid and a appropriate amount of insert for my ligation reactions, so the amount of plasmid needed to get colonies with this kit is less than 0.5 ng. That also means you always have tons of extra ligation left over in case something goes wrong.

On a side note using too much of your ligation can kill your transformation. We had a guy who couldn't get his cloning to work even though everything looked good. In the end it turned out he was using 10ul of the fast-link ligase reaction because that was what he did with a different kit in his old lab. He dropped the amount of ligation mix down to 1ul and it worked like a charm.

[jwb]

Thank you, monkfromork, for the reply!