Dear all,
I have transformed ZYCY10P3S2T E. coli (used for minicircle production, should be recA-) with following ligation mixture:
1. Insert: 3353 bp sequence containing gene of interest
2. Plasmid: pMC.BESPX_MCS2 (3904 bp, supplied by System Biosciences)
=> Total size: 7257 bp
After performing a transformation and subsequent miniprep, we ran a gel. When running the plasmid on a 1% agarose gel (TAE), 2 prominent bands appear for non-digested plasmid as you can see in the first two pictures.
- Picture 1:
- Lane 1: non-digested plasmid
- Lane 2: XbaI/BsmBI digested plasmid (4131 bp fragment and 3126 bp fragment)
- Lane 3 (pucture 1): 1kb Plus prestained ladder).
- Picture 2:
- Lane 1: non-digested plasmid
- Lane 2: XbaI digested plasmid
Is this what we see indeed a multimer/concatemer of our plasmid?
Nanopore sequencing by Plasmidsaurus looked completey fine, so is there anyone that could tell us whate we're seeing?
All the best,
Philip
First of all, I don't think ZYCY10P3S2T is RecA-, at least the one listing of the genotype did not show that it was.
Secondly, when you run uncut plasmid, most commonly you see a mixture of supercoiled and nicked plasmid, which will have differing mobilities. So a pure monomer prep will still normally show two bands from uncut plasmid that resolve into one band.
It seems to me that you are not seeing mulitmers, merely nicked and unnicked circle.
Thank you for your answer.
You are indeed correct, I misread the patent and they are indeed not recA-.
Most of the time, when I run undigested prepped plasmid, I get a band profile as shown in this picture.
But for this construct, the second band is always very bright. Any idea why it is way more present compared to other plasmids?
Dear Philip Shaw
It is common to observe more than one band in the undigested plasmid lane. These bands are separated as supercoiled plasmid < linear plasmid < nicked plasmid. Usually the supercoiled plasmid concentration is the highest in the sample, that's why you are observing thicker band on the lowest position. The highest band in your third picture might have arisen from the contamination of genomic DNA. I am hereby attaching two articles for better understanding.
https://blog.addgene.org/plasmids-101-dimers-and-multimers
https://bitesizebio.com/13524/how-to-identify-supercoils-nicks-and-circles-in-plasmid-preps/
Best wishes.
it is hard to say why your profile looks different from this strain. It may be something in strain that is causing a bit more nicking, perhaps some low level expression of mini circle creating enzymes.
In theory it could have meant that you have two different plasmids (ie two different supercoil sizes) but since you only see one size when digested, that rules that out.
Another possibility is that instead of supercoil and open circle, you are seeing supercoil form of monomer and dimer, which would always resolve out upon digestion.
I probably wouldn't worry much about it unless things aren't working for you.
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