I'm a sophomore (undergrad) at UTD and I'm working on transforming E. coli with certain plasmids. We have had some success using PCR, but are looking for a more efficient method. We are currently taking the plasmid and cutting it twice and attempting a "drop-in"? We added no primers to what was supposed to be our negative control, however we saw more colonies on our control plate than any others. We suspect that we may have had an incomplete digest of our plasmid to begin with and that background is what transformed into our competent cells for the control. However, we do not understand why we aren't seeing the same level of background on our other plates. Is it possible that the addition of the primers somehow prevented any uncut plasmids from transforming?
Many thanks!
Hi Freya,
I'm not sure I'm following what you are doing, maybe you can clarify:
1. "We are currently taking the plasmid and cutting it twice...", are you using the same restriction enzyme (RE) for the double cut or are you cutting with two different RE in the multiple cloning site?
2. "... attempting a "drop-in"... Is it possible that the addition of the primers somehow prevented any uncut plasmids from transforming?", not sure what you mean with a "drop-in", do you mean you are inserting something? If yes, you can't insert primers, you need a double-stranded DNA for the ligation reaction, so a PCR product, maybe? Digested like the plasmid? or you could of course anneal two primers designed to have overhangs (or not if you are cutting blunt with both REs) that fit to the cut plasmid...
lastly you should be ligating before transformation, aren't you?
Now, the reason for these questions is the following. In RE-based cloning you have two types of background: 1) uncut plasmid: depending on the enzyme you use, you might end up with even minimal amounts of completely uncut plasmid that you cannot see on a gel but that with competent cells that produce 10E8 colonies per microg of plasmid DNA will give you a number of colonies after transformation. For example 10 pg of DNA (10E-5 microg) will still produce 1000 colonies! 2) Single cut plasmid, meaning that on some plasmids only one of the two REs cuts, while the other doesn't get a chance, possibly due to kinetic (let's say time, although the meaning is "movement", but if you work too slowly time might not be enough!) reasons; in this case if you are ligating an insert it will have the chance to ligate to the single cut, but will not be able to close the plasmid ligating on the other RE cut, because on the plasmid it is missing (remember, we are supposing a single cut in the plasmid). So you see what happens, your control reaction without insert will efficiently close the single cut plasmid (intramolecular reaction, really quick and efficient) giving you a certain background, while in your reaction with the insert, the insert will ligate on one side of the single cut and will block the closing of the plasmid, meaning less colonies on your plate... Looks paradoxical, but it is a typical situation if you digest with two different REs. Seems to be your situation...
Keep in mind that if the RE is only one, independently from how many times it cuts, this situation cannot happen! Your insert will also have overhangs for the same RE cut and will always be able to close the plasmid, so you will not see appreciable differences between control and insertion experiments.
In addition to above suggestion you can also run a gel-electrophoresis after digestion running you cut plasmid, uncut plasmid and the insert you want to ligate. This not only clearifies wether you digestion was complete and give you a chance to purify plasmid and insert from the gel but you can also use it to look at the ratios you need when calculating your ligation. Of course these will only be rough estimates and compared to the concentration you probably use from nanopore...
Thank you Tim and Pietro! We are using two different restriction enzymes, and we are annealing two primers designed to have overhangs for the drop-in.
Ok, that's clear now. Then indeed you have one of the two REs that is not cutting efficiently... Could be old, could be degraded, could be bad quality (which Company?), or could be that it is not a good cutter (for example Kpn I has this problem, while the isoschizomer Asp718I cuts very well... also XhoI is not the best cutter, etc.)
Good luck!
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