Hi, 

You can try to see if the enzymes that you are using for digestion after miniprep are not digesting your vector backbone. We also had the same problem quite recently in our lab and we found that enzyme which we were using to digest was cutting the backbone (although it was not present in the MCS)!

Good luck!

I agree with Swati, you might have some extra recognition sites in the backbone. Take a backbone (without insert) and cut it with the same restriction enzyme(s) to see whether it yields the same extra bands.

Whatever the restriction enzyme I am using, it is giving the same extra band (see the gel).

You meant (from your last post) that the common band between 1,000 bp and 1,500 bp and some faint common bands, between 2,000-2,500 bp (in your picture), which present at each different enzyme-digested sample? 

Suggestion: grow a sample from the 'empty' competent cells (no vector electroporated; grow on the same medium without selection), and go through the same miniprep/and digestion procedures with the 'sample' colonies. To check whether these bands also present in the empty competent cells. If so, the competent cells might have been contaminated.

Hi there,

The extra bands you get sound like rRNAs (around 2.5kb and 1.3kb bands). Do you include RNAse in your resuspension buffer? I would suggest RNAse treatment at the end and see what happens... 

 Did you load the uncut clone also? Also digestion of the control plasmid will be helpful to analyse the results as others also suggested.

I agree with all above suggestion, Further better check the backbone and insert for restriction sites using online tools like NEBCutter, etc. We normally construct plasmids using VectorNTi software and one can also check the restriction sites using the same software. 

I feel may be you should use NEBcutter or any such software to check the appropriate restriction sites.

I think that most of the bands that you see between ~1,5 kb and ~ 3kb are uncut plasmid isoforms, since all these bands have a lower intensity then the band below 1,5 kb as the band intensity should be proportional to the fragment size if all fragments come from the same donor DNA. To be sure it would be helpful to know the fragment sizes that you expect for each digestion. Otherwise you could have a contamination in your preps which was already pointed out by Yuan. Good luck.

I agree with Thorsten. Considering the lower intensity these bands are most likely uncut plasmid. This is very common for DNA preps on cloning vectors, so might also occur for binary plasmids.

@ Eric,

But why the sizes are all the same across the different treatments (after different restriction digestions)? They shouldn't be the undigested 'whole' plasmid. Estimate from the gel result, the size of the whole plasmid is around 13.5 Kb. It should not have run that far !?

@  Dominique,

Regarding the suggestion of rRNA:

I think that RNA sizes are around 2.5 kb and 1.3 kb only when RNA size markers (single-stranded) are used for running and compared with the RNA fragments on a gel. I am not sure that the RNA sizes will maintain the same when they are run on a native TAE gel and measured with DNA size markers. I attached a picture below. It shows the total RNA isolated from bacteria (E. Coli). And, the RNA vs.DNA size markers are on the two sides of the picture.

The figure was taken from ZYMOTM Research website: http://www.zymoresearch.com/rna/total-rna-purification/bacterial-fungal-rna/zr-fungal-bacterial-rna-miniprep

Please read Yuan's answer carefully. It should help

would it be possible that you have genomic DNA contamination in your samples? Or maybe you have non-digested plasmid DNA?

@Yuan-Yeu Yau:

I think it's pretty easy and quick to add 1µL RNAse and incubate the samples prior running a new gel and see what happens instead of making bibliography on the RNA mobility on gel according electrophoresis system and ladder considered...

@Yuan-Yeu Yau:

Uncut plasmid can appear in different forms. Since these are all circular (and supercoiled) their sizes, ie running speed, will appear smaller than the linear plasmid size. Further, irrespective the enzymes used, the uncut forms will, since these remain uncut, run at the same size. When I started as PhD (1991) these ghostbands for cloning vectors were known as 'Freddy' and several technical papers were dedicated to that.

@Eric van der Graaff:

They will run lower, but a 14 kb plasmid will never be this supercoiled to run at 1.3 kb. At least i haven´t seen it, and i´ve been working with plasmids from 3 - 25 kb..

@Md. Mahmudul Hassan

I would also suggest it´s RNA contamination. Either try to add RNase or prolong the step in you plasmid preparation where RNA digestion occurs (normally the step after lysis and addition of binding buffer, where you´re told to put it on ice for 5 min).

Also be careful when you harvest your cells! This should be on the end of exponential phase, since during the exponential phase there is a lot more RNA in the cells than later on. But for E. coli 12 - 16 h at 37°C should be enough.

Good luck.

No, absolutely not RNA - the bands are way, way too sharp, especially for a non-denaturing gel.

The intensity of the lower band is telling. In equimolar concentrations (i.e.the band is not a doublet), smaller fragments in restriction digests bind less ethidium bromide and therefore fluoresce less intensely (unless ethidium bromide concentration in the lower portion of the gel is depleted because it migrates in the opposite direction of nucleic acids as it is positively charged). 

I had exactly this happen quite a while ago. Your issue is very likely to be the replicative form (RF) of a contaminating bacteriophage that co-purifies with your plasmid. And just like plasmids, the fainter larger bands are nicked/linearized forms of the ccc (covalently closed circular) RF form. This is not so common anymore, because many/most E. coli strains now have the tonA and tsx mutations that render the cell resistant to infection by several common, but certainly not all, bacteriophage. In my case, after wasting an entire month of graduate school trying to understand/get rid of it, I ended up ignoring it - in retrospect, it didn't affect my cloning. It was only several years later that I would understood the cause of the problem.

@ Dominique and Eric,

Thanks for responding to my questions to you.

@ Craig,

Thanks for the input. I am wondering: if that is the case (bacteria infected by bacteriophage), will the bacteriophage lyse away/ destroy the bacteria and affect the numbers of bacteria in the culture, which indirectly affects the amount of plasmids we want? I know you mentioned that they are 'Replicative form'. Will they ever switch to 'Lytic state'? If so, we should not ignore them, should we?

Lysis depends on the type of bacteriophage. For example, the T family phage (such as T4 and T7) is obligative lytic, lambda phage can switch between lysogen and lytic states, M13 phage does not cause lysis.

M13 RF can be grown/prepped as a plasmid and was commonly used as a cloning vector to generate single-stranded DNA (the form packaged in phage particles) for DNA sequencing.

I'm now thinking that that your issue could be a natural plasmid. Many common E. coli strains have been engineered to have a F' or F+ plasmid (fertility plasmid) to provide specialized functions such as conjugation or alpha/omega beta-gal complementation screening. I never really considered if the F plasmid would co-purify with a resistance plasmid (common antibiotic-resistant cloning plasmid) during a plasmid prep - maybe someone on RG knows? I would expect F plasmids to naturally be very low copy number though, but I could be wrong.

p.s. just checked - your ElectroSHOX Competent Cells genotype is F- (no F plasmid), but they are not tonA tsx (phage resistant). Try scraping a few cells from the top of a frozen vial of competent cells (untransformed) and inoculate a few mLs of L broth for a miniprep. Run on a gel to see if your extra band comes from the cells.

@ Craig,

It is an interesting discussion.

1. So, in summary, from your point of views, there are two possibility for the extra bands: (1) natural plasmids in the bacteria (such as F+ plasmid, apart from the 'standard' cloning plasmid), and (2) possible bacteriophage infection.

2. The genotype of the competent cells, ElectroSHOX Competent Cells, Md. Mahmudul Hassan is using is:  F- (no F plasmid), but they are not tonA tsx (phage resistant). So, the extra bands were more likely from bacteriophage infection than from the natural F plasmid.

I think that it makes more sense taht the F- genotype is used, because F+ plasmids can integrate into bacterial chromosomes and result in bacterial phenotypes, unless there is a specific site for them to integrate.

3. Regarding your question: "I never really considered if the F plasmid would co-purify with a resistance plasmid (common antibiotic-resistant cloning plasmid) during a plasmid prep - maybe someone on RG knows?"

I found a similar discussion on RG**: "Will the F' plasmid in OmniMAX 2 cells be co-purified after transformation of pDONR?" 

See Dr. Johann Heider's response: "The answer is that F has such a low copy number and is so much larger than "standard" plasmids that it is always expected to be out-competed in transformations. Most of it will probably also be entangled enough with chromosomal DNA to be separated from the (small) plasmid fraction during plasmid prep."

**https://www.researchgate.net/post/Will_the_F_plasmid_in_OmniMAX_2_cells_be_co-purified_after_transformation_of_pDONR

Thank you very much Craig M Neville, Yuan-Yeu Yau and all other for your nice comments. It was a great discussion and I have learned a lot from this discussion. @Craig M Neville and Yuan-Yeu Yau, For your information, I have only found this unexpected band when I used this particular E. coli strain. Also when I tried to introduce the construct (one that has the unexpected 2.5 and 1.2 kb additional band), I did not get any colony in my plate although positive control worked. It seemed that this additional band somehow causing problem in Agro transformation. Now, I have collected a bunch of E. coli strains which are known to handle very unstable construct (as my plasmid is also a unstable one). I will start the cloning next week again and will let you know what happens. Thank you all very much again.

@ Md. Mahmudul Hassan,

Thanks for updating!

1. Indeed, it was a great discussion. Thanks for posting this question and updating us. If you could, you should bring this issue to the company's attention, and see what they are going to say.

2. Intra-plasmid DNA rearrangement has been reported. Especially those constructs with repeats. I attached a paper for your reference (see below). In your another question, you mentioned that you are using Top10, NEB stable beta competent cell for your unstable plasmids. They are specific to be used for reducing 'recombination' (ex. between repeats of a construct). They have a recA1 genotype to reduce recombination of cloned DNA. Hopefully, you will get what you want.

Checkout below preprint, Hopefully you may get some answer out of it

RNA interference in gene cloning/expression

https://peerj.com/preprints/1831/

Hi ,

I would like to suggest sth very simple,

It is quite common to see multiple bands within a single lane when a plasmid sample is run on an agarose gel. These bands represent different forms of the plasmid. Usually, one of the bands is clearly predominant and this is the supercoiled form of the plasmid. Usually, there are 1-3 bands above the supercoil band, indicating plasmid species with slower electrophoretic mobility. These are usually the nicked and dimeric forms of the plasmid (or different combinations thereof). Occasionally, there may be a faint band which runs slightly ahead of the supercoil. This is referred to as the “irreversibly denatured” plasmid and is a biproduct of alkaline lysis. This form of the plasmid is refractory to many/most enzymatic reactions, including restriction and sequencing.

This is in the case of an intact plasmid. For your case, i suggest you to run your intact undigested plasmid once along with your other samples and observe the bands. depending on the enzyme you're using for digestion and the time you keep your samples for digestion, it is possible that still there are some undigested plasmids present in the solution you load on the gel. 

I hope you find this helpful :)