Failed cloning for 1month - TOPO - (Apr/05/2014 )

I have been trying to clone an insert into a vector for 1month now and gotten NO RESULTS!

Initially, it was the standard ligation-approach, but then we decided to use the more simpler TOPO cloning approach.

Now I have tried TOPO two times and still getting no colonies.

The PCR of my insert gives me strong clean bands on the agaros electrophoresis. As a backup I did two approaches.

1) I ran a PCR-sample of total volume 50ul. Of this I loaded 5ul on a electrophoresis and got a nice clean band. Thereafter I purified the remaining 45ul (ThermoScientific PCR Purification Kit) and got a final DNA-conenctration of 150ng/ul.

I used 1ul of this for my TOPO-cloning (incubated 10min, RT). Then transformed into DH5-cells. Incubated overnight.

The ONLY way you can make progress is by starting to do controls. You can start by incubating some empty plates, and see if there is growth of colonies after 48 hours.

So, the first TOPO cloning was made by creating overhangs on a Phusion-generated PCR-template using DreamTaq. And that experiment did not give any colonies at all. Presumambly because there were no overhangs created.

I did the TOPO again the day after with a freshly generated PCR-template directly amplified with DreamTaq (and not Phusion, thus no need to create the overhangs later on). And today, I got colonies. So, now I will harvest these colonies in LB-antibiotic media for overnight growth, and tomorrow do the mini-prepp followed by EcoR1-digestion and final sequencing.

Question)

My PCR-template had no antibiotic-resistancy in the sequence. After cloning it into the TOPO-vector, it now carries resistance for both Ampicilin and Kanamycin. And I have gotten growth on both of these plates (as expected). But how should I go on from here? Harvest bacteria and do the miniprepp/digestion with only the colonies from the ampicilin-plate? Or the Kanamycin-plate? Or just do miniprepp/digestion on both separate ones?

phage434 on Sat Apr 5 12:32:18 2014 said:

The ONLY way you can make progress is by starting to do controls. You can start by incubating some empty plates, and see if there is growth of colonies after 48 hours.

If your plasmid has resistance to two antibiotics, it would be best to grow it with both, which will assure both resistance genes are functional. Colonies from either plate should grow in this double antibiotic medium. You should grow and miniprep several colonies (perhaps two from each type of plate). Then digest with an enzyme which will release the insert, and run the results on a gel to verify that you have created the correct plasmid.

On TOPO vectors I use kanamycin whenever possible. It's more stable and therefore less prone to overgrowth. Unless there are some problems with overgrowth on the sole kanamycin I woudn't see a reason why to use two antibiotics.

Of course when intact and functional amplicilin resistance gene is required later, using both may have sense. But for normal plasmid propagation I woudn't do that, I take ampicilin resistence gene there only as a bonus I don't need to use.

(and, when I think of colonies only growing on an ampicilin plate after 48 hours, I would't trust them at all, ampicilin is thermaly unstable, degrades after prolonged incubation on 37, that's also a reason why I prefer kanamycin when possible or use the higher recommended concentrations of amp in medium)

I also avoid amp during design. If i need to grow an amp resistant plasmid, I will use carbenicillin instead of amp, but I try to avoid even that.

phage434 on Sun Apr 6 13:20:23 2014 said:

I also avoid amp during design. If i need to grow an amp resistant plasmid, I will use carbenicillin instead of amp, but I try to avoid even that.

why?

Amp is used on a routine basis in many research subjects so I wonder why you don't want to use it. 

I can understand the problems with the beta lactamase  but is this such a big issue? (if yes, than why do so many people still use this amp?)

Ampicilin gets degraded quickly in higher (cultivation) temperatures (also can't be stored in plates as long as kanamycin for instability reason) and bacteria can secret beta-lactamase into the enviroment.

You get more overgrowth in ampicilin media in general and more problems with satellite colonies. And can get worse when handled bad (you add to medium that is too hot, use old plates etc.).That are just two serious reasons why ampicilin just sucks. 

The main reason, why many people have to use it still I think (and by the way no one I know would choose amp over kan), is simply because they have old plasmids (or) with amp resistance only. Or of course for combination, if you co-transfect/need to select on kanamycin background. The other antibiotics are even less widedspread.