Dear Niklas,

All three plasmids have pUC origin of replication, as the like you provided mentions. They also have Amp resistant selection markers. I already transformed mixed solution into E.coli. Trasformants have also observed. But what is worrying me is if there are all three plasmids (5.5Kb, 5.9Kb, and 11.5Kb) in same colonies. Additionally, I have extracted plasmid DNA and observed only two bands, As all plasmid maps are the same, meaning same markers and Ori, as well as same transformed colonies on agar, I do not know how to select each one of them.

Hi,

I didn't understand if you want the three plasmids in one bacteria or each plasmid in a different one. It will be easier to a have three of them together if each one contains a different resistance cassette.

In a "normal" agarose gel, you will see few band for one plasmid extracted (because of the differents states - relaxed / coiled / super-coiled ...), so to see two bands isn't imply that you have two differents plasmids. But maybe you are using some particular gel or, your plasmids were linearized before.

On your plasmids extraction, you can do a restriction with an enzyme that can cut the three plasmids you have. If it is work, you will see bands of differents sizes.

Maybe it will be easier to begin again and do one transformation each time.

Good luck

Dear Laure,

I want them all in one bacteria as they have same resistance marker, Amp. So you are saying different restriction enzymes may give me three different bands with different sizes. Sounds right and very helpful. I will upload my digestion results here.

Thank you so much.

Alireza,

You will not get the three plasmids in the same cell. Even ignoring the fact that the odds of co-transformation with all three plasmids are low, they all share the same selection marker and replicon (hence are incompatible), so there is no way you can select for a cell containing the three of them.

If you do enough minipreps, you will find colonies bearing each of the three plasmids (you do have a means to tell them apart, do you?). All you have to do is purify them separately and mix them whenever you want to prepare the packaging mix.

Hope it helps!

Dear Dr. Martin,

As you said all three plasmids have the same marker, for which after transformation into competent bacteria, I have chosen all transformed colonies for maxipreps. Unfortunately, I recently graduated and have no access into a lab for further examinations. However, plasmids have their unique size and I suppose the maxipreps DNA extractions may have all three plasmids. If you are agree and there are no further barriers I will digest 1ug of maxiprep with certain endonucleases.

Sincerely