Are these all different clones of the same plasmid? Or different plasmids that would be expected to have different banding patterns?

I have 2 thoughts:

1) Star activity. Restriction enzymes aren't as ultra stringent as we tend to think and if your plasmid has pseudo-restriction sites (i.e. sites that differ by only 1bp from the canonical restriction sequence), then you can get non-specific cutting at those spots in reactions with too much enzyme or that have been incubated too long. This non-specific activity may account for the presence of unexpected bands

2) Incomplete digestion. Counterintuitively, an alternative explanation might be the opposite of the above. If you're not getting 100% digestion of each enzyme in that particular reaction (maybe because of carryover contaminants in your input DNA or some issue with temperature), then you'll see other distinct DNA species arising from uncut, single-cut, double-cut, etc. molecules. This could fit because some of your unexpected bands look like they're larger than the main diagnostic bands in the other samples.

I'd recommend the following:

A) Run single-enzyme controls with verified DNA in parallel to each of these diagnostic digests. This will allow you to assess if your enzymes are working as expected.

B) Decrease the volume of DNA going into the digestion reaction (keep it to

Hey Govinda, thanks for taking the time to answer. These are all different plasmids and the band patterns are correct but for a couple of faint bands that I'm pretty sure are due to incomplete BamHI digestion, and BamHI can be sensitive to high salt. I did discover that I accidentally added twice as much buffer as I should have, but all samples were treated the same so that would not account for the difference between samples. At this point I am debating an ethanol precipitation vs a re-purification of the plasmid in question (lane 6).

Makes sense. BamHI is famously also one of those ones that will cut non specifically if you let it.

I think you just have genomic DNA contamination in the one sample with the fuzzy background. It is of course DNA so the A260/280 ratio will look fine, but when digested by restriction enzyme you get a multitude of randomly sized bands (hence the smear). If you were using less DNA in your samples you might not even notice it, and if you used more DNA in the other samples you might begin to see more background in those.

I would include a control with buffer only in the reaction. Adding 2x buffer could be a problem

If it's an option, New England Biolabs now sells a high fidelity version of BamHI (BamHI-HFv2 or something like that). It's supposed to have much better specificity and activity than the original BamHI (which is one finicky enzyme!)

Okay so new update, I reran the plasmid in lane 6 without restriction enzyme and got the result below. Lane 1 is NEB 1kb ladder, Lane 2 is a control plasmid I know is correct, and Lane 3 is the same plasmid as in Lane 6 above. So I still don't know what's going on here except maybe degradation. I retransformed with the plasmid in question to try to purify a correct clone since there does seem to be some plasmid remaining, but my first purification yielded the wrong plasmid. I am now screening 12 more and hopefully one will be good enough to Maxiprep later.

So just out of curiosity...what do y'all think happened here? Just standard degradation?

Maybe some salt and/or degradation. A new prep would help with either problem. When you say the first purification yielded the wrong plasmid, what do you mean? Wrong band pattern/sizes? Too much smearing?

Wrong band pattern. I just screened 12 more colonies and they all have the same band pattern, all incorrect - approximately 1200 bp is missing. This is so weird, considering that the dominant band pattern in the digest of my streaky parent plasmid is correct. Only thing I can think of is that my desired plasmid is at a competitive disadvantage with a mutated or contaminating plasmid. Looks like I may have to remake it.

I'm presuming that the plasmid is correct when initially transforming i.e. contain the 1200bp section?

Then after transformation you lose this section?

If then your plasmid is unstable in the strain you are using. What bacterial strain do you transform into?

There can be many reasons why a plasmid is unstable, something in your sequence such a repeating sequence for example. Try using a different strain to transform, recA- etc or if it is repeats use a strain that can cope with this. It might be that you are selecting for colonies that are faster growing as they have lost a portion of the insert over those that retain full insert and grow slower. I had this problem with a particular gene insert in which colonies that harboured the correct plasmid (intact) grew much slower..so I grew plates a little longer and took the small colonies to prep and luckily it worked.

I cant however explain your gel as it looks like selective degradation of the lower band?

Did you check with the sequence for existence of known BamHI sites or was this insert new (new strain, variety) The missing fragment could bedue to another missed (misinterpreted absence of extra) site. try with linearization of the plasmid with one unique restriction site and compare with the vactor in next lane along side a ladder lane (1200 bp can be justified, in case all your 12 clones of the same construct were lacking the 1200 bps). Just a case may be. OR sometimes I feel if this is your expression vector, might be the bacterial host hates it (toxicity issues and does good to itself cleaving it, dont know if I am right, but might be at least for larger BACmids with toxic inserts and the DH10Bs always altering the restriction patterns for me while I was making some CoV constructs back during my doctoral tenures). Science and serendipity, you see...

I'm a bit late to the discussion, so apologies if you've covered this, but maybe you could run a control on the gel of the plasmid that's smearing where you have not digested it first (that would help you tell if the smears are due to BamHI cutting indiscriminately, or because of how you're preparing your plasmid DNA).

Just as a final update, I decided chasing my tail on this wasn't worth my time and I just recloned the plasmid. ¯\_(ツ)_/¯

What E. coli strain are you using as host?

Stellar

Just checking to be sure it was an EndA- strain, but Stellar is. Did you at least just try to make a new plasmid prep before recloning your insert?

Yeah, after retransforming and checking 12 different colonies, all 12 colonies were wrong. Something weird happened and I just lost my insert so that's why I recloned it.