Anyone with experience with cloning using E.coli DH5Alfa and Pjet 1.2 plasmid who can share their experience with me? I've been trying to understand and identify if my clones are showing DNA supercoiling, but it's been very difficult to find out about it. Plasmid linearization is a rare subject to find (at least where I looked). Thank you in advance for your contributions.
Dear Elis,
I am not sure if I understand your problem clearly, but you may have got yourself a little confused about the advantages of using a cloning vector such as pJET1.2.
In essence, these vectors are supplied in a form that allows immediate cloning – it has already been linearised and will accept a DNA fragment which is blunt-ended. You then use this to transform your host cells, such as DH5alpha, and select transformant colonies on agar plates containing ampicillin. This should be very effective, as if the DNA fragment has been cloned into the vector, the lethal gene will be deactivated and the host cells will not die.
You should isolate DNA from several transformant colonies. If you use a mini-prep–type system, it is likely that most of the plasmid DNA you isolate will be supercoiled (more impure DNA will have more uncoiled/relaxed DNA, and perhaps even some linear and degraded DNA).
It is probably better to examine the transformant colony plasmid DNA by gel electrophoresis after digestion with restriction enzymes which would (i) linearise the plasmid, making it much easier to determine fragment sizes, and (ii) confirm the presence and size of the cloned fragment. Of course, it may be faster and easier to confirm the presence of the insert using a PCR approach, and for this it does not matter if your plasmid DNA is supercoiled or not.
Regards,
Andrew
Dear André, first thanks for the reply. Second sorry for the confusion I made for understanding. So, I did the whole cloning process that you've already described above, and the extraction of the clones I did with an in-house mini-prep that we use. After that, I did PCR screening in 1% agarose. The first time two bands were formed in some clones. Another detail I noticed is that the bacteria of these clones were mucoid and quite large. In this way, I made a selection of 2 clones that the agarose gel band did not "drag" and I made a new cloning. In this second cloning I obtained colonies of totally different bacteria in smaller quantities and dry and frosted. After the cloning and extraction process, I re-PCR screened the selected clones and obtained a single-band PCR for each clone in the run. I went to try to understand and ended up reading about supercoiling of plasmids and I confess that it has been difficult to find literature on understanding the subject well. My main doubt is whether the clones that I am going to use can have their quantification altered in the Qubit device due to this supercoiling? This would affect my quantitative real-time PCR. Thank you very much
Elis Regina Alves Pinto , you should just not worry about supercoiling for your conditions. First of all when you isolate plasmid DNA it will be predominantly supercoiled if you do not digest it. Upon digestion with an RE or upon nicking you convert it entirely to either linear or open circle form.
However all of the above templates will work fine for PCR.
Regarding the QUBIT measurement, it won't make that big a difference. I think in theory there might be 20-30% difference between fully supercoiled and non-supercoiled plasmid. Most mini preps are not going to be 100% supercoiled, so the difference would be even less. This difference is due to how much dye the DNA can bind if supercoiled.
Thanks, Michael...I feel more comfortable knowing this information. thanks for listening.
- Badges
- Science topic