Hi Emma Thompson

Actually, there are three different structures of well extracted plasmid：Linear, circular and super spiral. The migration rate in gel is circular〉super spiral〉Linear. Therefore, the expected size will be larger after double or single digest naturally (All are linear structures)

• If the present band size really larger or smaller too much than you expection, that makes no sense.
• If there is any possible that the backbone plasmid which you received form other lab has some genes insertion (double check).

Emma Thompson as mentioned by Beibei Zhang, you can get different plasmid mobilities from uncut plasmid. I would normally not rely on uncut except perhaps for quick screening a number of putative clones. And even then you need all the right controls.

Give your uncertainty I would either 1) do a digestion that should release the insert from the plasmid and see if those both have the appropriate sizes

or 2) do a PCR of your insert to be sure it is present and of the right size.

It is possible that the plasmid stock you received had a mixture of plasmid only and plasmid with insert and maybe you just picked the wrong colony. If the above analysis show there is a problem then I would retransform from the original DNA stock you received and screen a few transformants in the same way.

Emma Thompson Michael J. Benedik i agree with Dr. Michael J. Benedik. Based on the theory, the Escherichia coli clone competent cell is just a host for the propagation of your recombinant plasmid, and has minim chance of recombination with host genome. So the possible of your fasle results maybe because of your original plasmid or your inproper selection of positive clones, and can be fixed as mentioned by Dr. Michael J. Benedik.

Beibei Zhang Michael J. Benedik Thank you for your responses, though I'm not sure they contributed towards an answer - perhaps I phrased my question incorrectly. I'm aware plasmid mobility varies according to plasmid topology which is why I included details of size according to whether supercoiled vs linear.

My confusion stems from the grossly unexpected size ascertained from the agarose gels: too low in the case of the commercially acquired pGenDONR_mygene (despite linearisation) and too high in the case of pPICZa-DEST which was maxing out the ladder in the supercoiled form. However, since I posted the original question I ran a gel of linearised (single digest) pPICZa-DEST in which the size drastically dropped - suggesting the plasmid was concatenated. In regard to pGenDONR_mygene, I am going back to square one and transforming E coli TOP10 again. I will pick more than just a few colonies this time. If I'm still seeing the same problem, I will contact Genscript as this was a custom gene synthesis.

I should also add:

I would expect to see the supercoiled form of pPICZa-DEST (5300 bp vector) at ~3700 bp according to the calculation which assumes that supercoiled DNA runs ~30% faster than linear. (5300 x 0.7 = 3700). But instead, I see supercoiled/uncut form at ~10,000 bp!

Emma Thompson I just realized that you are preparing for yeast transformation (protein expression). I dis this before, and met the same situation like you. The expection size will become smaller drastically indeed, but the target protein of my own in the supernatant of yeast was not affected actually.

So i have some sugestions

• Do the PCR with your original recombinant plasmid as template, to confirm the presense of your target gene, and change another compontent cells like DH5α, XL1-Blue or JM109 etc.
• Select three positive clones and propagate in 200mL BL medium overnight, then conduct the DNA plasmid extraction with your commerical kit

• 10~15ug linearized plasmid is necessary for the ensurment of positive rate after transformation

Emma Thompson

Do not care about the size of your plasmid too much if you confirm the prense of your target gene with PCR. Change the competent cells and conduct the linearization and transformation of yeast. The positive yeast clones with your gene should be checked again indirectly though SDS-PAGE of translation products in the yeast supernatant

I have quite a few times run into a plasmid dimer that is "locked" in the dimer form since the host strain is RecA-. This would be a concatamer (and not a concatenate). Upon digestion you would observe the monomer size.

Regardless, retransformation and analysis of some new transformants should hopefully give you want you are looking for.