Filippo Rossignoli Make sure the competent bacterial cells (ElectroMAX™ DH10B Cells) doesn't contaminated with other plasmid before you use them. Do a negative control (competent cells without electroplating plasmids), and spread it on your plate, to see if they are growing.

You may increase the concentration of antibiotic in the media to avoid false-positive colonies. Try to sterilize laminar airflow, all the tip boxes, pipettes, etc make sure no chance of contamination. While incubating plates, make sure they are sealed with parafilm on sides. Hope it helps!!

There are some light larger band visible to me in lane 2-10. It seems that the gel electrophoresis needs to be continued for some more time. There is chance that running for longer cause the band disappear because the concentration of the DNA looks very low in these band. Large plasmids are low copy number plasmids, i am not sure about copy number of a lentiviral plasmid . So you need to increase the number of the plasmid by increasing selective pressure and optimize miniprep.

Another thing you can do in order to verify that the colonies contain your plasmid of interest is using colony PCR. If you have the sequence of your plasmid, you can design specific primers that will lead to PCR product only in the presence of your plasmid.

For more details you can use this link: https://international.neb.com/applications/cloning-and-synthetic-biology/dna-analysis/colony-pcr

Good luck!

Could the small plasmid you are seeing be from the source of the insert you are trying to clone? But you need to do a set of controls with your transformation such as no DNA, insert only, vector only, and maybe ligase + buffer only to see which is the source of transformants.

I agree with Shaista. You have in all your samples plasmid running as your control.

How did your PCR looked like and also after digestion?

are you sure that your minipreps are effectively cut ? the bands you observed could be uncut suppercoiled plasmid: try another restriction enzyme that should give you several fragments with , if possible, at least one that has a different length that the parental plasmids. you then will be sure your minipreps are cleaved and that the clones are from your parental plasmids or the one you want .

It is possible that your digestion is not optimal. Please share the details of the digestion protocol as well. Additionally, is the plasmid in the first lane digested ? Because from the gel images, it seems that there is a faint band at your desired size and a strong band below which might be due to supercoiled conformation. Best way to check is to run a PCR for your gene taking the isolated plasmid as template.

I also see a faint, large band in all of your samples, except for the one closest to the bright band. The smaller band could be supercoiled (as others have said) or it could be a digested product that has your insert. How many times does the restriction enzyme cut in your plasmid + insert? If there are two cut sites, then you are seeing a product from the digest.

Yuan-Yeu Yau thanks for the suggestion. The batch of bacteria is new every time and nothing grows if plated untransformed

Gopal Tiwari Thanks! I'll try to sterilize everything more accurately and try again. I already doubled ampicillin concentration without any improvement. Two plates grown at the same time, one with parafilm and one without, gave similar colonies.

Shaista Bano Thank you. I tried to increase ampicillin without success. Also miniprep with resin based column kits don't change the outcome :(

Oren Bachar thank you. It doesn't solve the issue but it will help finding the right colony so I'll give it a shot!

Michael J. Benedik Thanks! The insert comes from a pcr amplified product which is digested and gel-purified, so not directly from a plasmid. I also tried all the control you mention and I have colonies only if transforming with backbone only or ligation product

Jana Schmitzova thank you. PCR looked fine. A single neat band. Same after digestion.

Didier Poncet Thanks! I understand your point but the expected size of the plasmid is >10Kb (as you can see in the first lane) so I don't expect it to migrate so much even in supercoiled form. I tried to digest these kind of plasmids without success.

Jatinder Chera Thank you! My digestion is 300-500ng plasmid, 5U of restriction enzyme, buffer and water. 1h at 37°C. I agree there is a faint band at the desired size, I'll try a PCR to screen. I'm not convinced about the supercoiled conformation cause it's way too low. The first lane is another plasmid (undigested) with comparable size and doesn't migrate that far in the gel. Also I tried to digest these 3Kb plasmids without success.

Katie A Burnette Thank you! The gel I show here was loaded with undigested plasmid, so it might be I have a mix of two kinds of bacteria, given the faint band on the top.

Filippo - two other thoughts. First run some of the digested vector on a gel to see if perhaps there is a problem with the vector (or vector prep) generating that smaller band you see. Secondly you say that you get cookies if transforming only the backbone - have you looked at those to see if you are getting small bands or only the correct size. You might even do a mock experiment where you digest and relegate the backbone only and also the PCR product only and transform them to see what you get from those controls. If you think through it logically you either are introducing a contaminant plasmid from somewhere or your digestion and ligation is generating an unexpected plasmid.

You can screen your colonies with a colony PCR, which is way less work than mini prepping all the plasmids. Make a numbered grid on the back of an agar plate, and put a little of each colony in its own square, and grow the plate overnight. Now you have numbered small streaks of bacteria. Run a PCR with primers you already have, preferably one in the gene of interest and one outside it, but your cloning primers are fine. Use a little bit of bacteria as the template. A boil step for a few min in 10 ul of water before addition of the other reagents works well to release the DNA from the bacteria. If the PCR product is the right size, then you have a good chance of having the right plasmid. The ones with the right size product can be mini prepped and sequenced.

Hi there,

I see gel-purification for the PCR amplified insert. Do you also gel-purify and dephosphorylate your digested vector?

And do you add ligation control without insert to have an idea of recircularization and false positives?

Definitely screen your clones with a backup on a cases on a plate and PCR on the rest, it'll save you a lot of time.

Best

Daniel

Filippo Rossignoli Try setting up a digest of your empty vector vs. plasmids that possibly have the insert. This time, pick an enzyme that you KNOW will cut the empty vector more than once. Should be easy to do with online tools and/or the plasmid map. Write down the exact expected product sizes for both the empty vector & the vector with the insert (don't forget to check the insert for digest sites). Basically, make your own digest maps.

Set up larger-volume digests. Use MORE plasmid DNA for the digests. Remember, these are large molecules so you need to use more DNA to have enough to see on a gel, especially once it's cut into pieces.

Pour a gel using the widest gel combs you have, load ALL of the digest into the gel. Include: DNA ladder, undigested empty vector, digested empty vector, undigested plasmids with possible inserts, digested plasmids with possible insert, your original PCR insert (if you have it).

Time to go "old school" and get it sorted out!

Good luck!

Hello everyone!

Thank you for all your inputs.

In the end we were unable to understand what was going on, so we decided to switch from the ElectroMAX™ DH10B Cells to the chemical competent Stbl3.

We transformed the ligation reaction (performed with electroligase) into the Stbl3 by heat shock and we got few correct colonies and no "contaminating" plasmids.

This doesn't explain what we were seeing but at least the cloning is now done!

Thank you all again.

Filippo