Dear all,
I am facing a problem with plasmid isolation from thermophilic bacteria. For one strain I was getting one single band of 1000 bp using Thermo Scientific GeneJET Plasmid Miniprep Kit and same result using a modified manual alkaline lysis method (I get the same result for more than 20 times, the same size of band). I was thinking that it’s a plasmid even getting one band not 3 or 4 bands. But after double digestion using Hind3 & BamH1 and clonig into puc18, the sequencing result reveal that its a fragment from genomic DNA !! and the strange that i didnt found the vector sequence using VecScreen ? I am so confused about result, its just my mistake during ligation or digestion or its not a plasmid !!!
Do you have any ideas, please?
It would help to see a picture. But if you are doing a normal mini prep and you are getting a 1kb band then it likely is not genomic DNA, but either a plasmid or a phage of some type. Perhaps the fragment you cloned is not actually from the 1000 bp band but is from some contaminating genomic DNA.
Did you test to show that HindIII and BamHI both digest this 1000 bp fragment? Statistically speaking it is possible but unlikely.
1000 bp is a bit small for a plasmid, but it's possible. Especially since supercoiled plasmids move faster than their linear size. Did you run out any of your digest on a gel? I'd start by looking for evidence of a successful restriction digest. Include a positive control plasmid (something you KNOW is cut by Hind3, BamH1, or both). My guess is that either your plasmid isn't cut by those enzymes or your digest didn't work and your cloning reaction picked up a few fragments of genomic DNA that were carried over in the reaction. Good luck!
Michael J. Benedik , Thank you for reply, you find attached some pictures, yes I have check double digestion result, it was good, and after cutting of my clones (plasmid from white colonies) I get a band of the same size of my insert. Please what do you suggest to confirm if it's a plasmid or something else ???
My best regards
Katie A Burnette Thank you so much for your reply, after the first digestion with Hind 3, I got a single band of 1500 bp (good digestion), but using BamH1 I got almost a band of 1600 bp (so I am confused regarding Bam H1 digestion), maybe I should repeat this step again.
The size difference can be due to supercoiling (positive or negative) compared to relaxed, nicked, or linearized forms of a plasmid. The salt content of a digest can change how a circular plasmid moves on an agarose gel. Remember, only linearized DNA molecules move at the same way as your DNA ladder. Circular DNA can run "too fast" or "too slow" due to their shape.
My guess is that your plasmid isn't being cut by these restriction enzymes.
It is difficult to determine what is really happening. I think Katie A Burnette is probably correct that you are just seeing some nicking of your supercoiled plasmid and converting supercoil to open circle. It may be that HindIII is digesting but I don't think the other enzymes are. You might try to ligate after only HindIII digestion and see if you get anything.
Michael J. Benedik I have already try to ligate after only HindII digestion, but Transformation efficiency was so low (2 or 3 white colonies), and it was not cloned, this is why I tried this time double digestion.
Katie A Burnette, so you think even with HindIII its not a cutting ?? any suggestion for real digestion, and how can I have a plasmid with 3 or 4 bands in the same time, because until now I've got one single band (seems like circular or supercoloid)
Why don't you try a 4 bp cuter -Sau3A I or Alu I for instance- and clone/sequence some of the largest resulting fragments to see what you've got?
Alejandro Martin Thank you for reply, yes I have already try digestion using Sau3A I, unfortunately plasmid was completly digsted even with smll amout of enzyme (6U/µl) for just 5min (I try befor same concentration for 1h and 30 min same result : complet digestion : I found nothing on gel)
That is not normal; if you really had a 1-1.5 kb plasmid in there it would have yielded a discrete number of fragments. Try repeating the digestion, but loading 5 times as much DNA on the gel (the mass of each individual fragment will be much lower than that of the plasmid) and using higher percentage agarose to account for the increased relative mobility of the resulting fragments.
Leyla Benammar It's possible that the HindIII doesn't cut this plasmid. Did you use a positive control reaction? I'd want to check if my enzyme is still working since it's more likely that either your enzyme or buffer has expired than not having a HindIII restriction site in 1000 bp. Still, it's possible.
For your 4 base-cutter, you could try using it for a very short time in a sub-optimal buffer. According to NEB, their Sau3AI enzyme has only 10% activity in buffer 3.1
Good luck!
Katie A Burnette yes I have tried HindIII with other samples and it works, even for my friend in lab no problem detected. For Sau3AI I have tried 1.1 buffer 100% activity. I will try 3.1 buffer I hope it works this time. Thank you so much
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