Hi all,
I am trying to extract natural plasmids from my bacterial strains. I don't know the size of plasmids, nor if different plasmids are present in the same strain. I run a gel with the plasmidic extract and I detected some bands. I was suggested to do a restriction digestion (with different enzymes, I used three )of the plasmidic extract and run the gel again to determine the size of the plasmid. But, after the digestion , no bands are present on the gel, not even the uncut DNA . This happens for all the three enzymes used. What could it mean for you? What could it have been wrong?Can you also suggest me how can I determine if different types of plasmids and how many copies of each are present? Thanks
Hi,
I think your restriction have worked.
From your description the guesses could be:
1. Your enzymes could be a most frequent cutter.
2. Check gel percentage.
Check the incubation time and other parameters like amount of enzyme added were followed as per standards
Hello Silvia,
I would agree with Krishna Raja regarding the enzymes being frequent cutters and thereby totally degrading the plasmid. Well, I would recommend that you send your purified plasmid DNA for sequencing with commercially available primers so you can have a map. Then its easier to study what enzyme modifications do to the plasmid.
If you do not prefer to sequence, then probably try some restriction enzymes with very low star activity and very rare restriction site (NotI, Bbv CI....check for enzymes with restriction site 6-8bp long...https://www.neb.com/tools-and-resources/selection-charts/alphabetized-list-of-recognition-specificities).
Now as for your undigested DNA also vanishing, I think it could possibly have to do with gel composition, maybe try using a higher % gel to run your samples. Also.....if you were using a DNA ladder, where was its location when you finished your run? It is advisable at first to run the gel till the ladder is nearly resolved and to avoid excess separation. This is because, you get an idea where your product is and accordingly you can re-run the gel to resolve it better. However, a long run can cause you to run the product out especially if you are unaware of its Molecular weight.
Hope this helps.
Regards
I think that your plasmid isolation contains some DNAses that are eating your DNA. This happens at 37ºC, when you are performing your restriction digestions, due to this, at the end you don´t have DNA at all. Maybe you need to improve your DNA isolation protocol, for example by treating your DNA solution with Phenol/chloroform in order to get rid of DNAses existing in your DNA preparation. But after this, you should clean away from your DNA solution all the phenol traces, because your restriction enzimes will not work with them. You should perform a precipitation of the DNA (with acid pH and cold ethanol) and afterwards clean with cold 70% Ethanol to clear away the salts of the DNA solution.
Good luck!!
Hi,
One more suggestion could be performing a control restriction reaction along with your test reaction. This positive control can be any known plasmid or commercially available vectors used in your lab for routine purpose. Since you know the size of the restricted products this gel will help to point out where the error could be.
Good Luck!
If you don't know what plasmid(s) are in your bacterial strain, then how are you maintaining the plasmid in culture? Unless you're treating the cells with one or more antibiotics, it is likely that you will lose them. Depending on what plasmid isolation protocol you used, the bands that you saw may have been genomic DNA purified at a low level (if the plasmid extraction was not performed fast enough), which would become dilute and difficult to see on a gel after adding to a restriction digest.
What size(s) were the band(s) you saw on the gel? Remember that purified plasmid DNA will run at 2-3 molecular weights, corresponding to supercoiled, nicked, and linear DNA (i.e. the multiple bands you saw may not have been different plasmids, just different topologies of the same plasmid).
As mentioned above, it is possible that you are using common cutters that essentially degrade the plasmid. BamHI or HindIII are usually good for generating one or more easily-visible products on a gel. Whichever enzyme you chose to use, remember to never let the enzyme concentration be less than 1/10th of the reaction volume (otherwise the glycerol in the storage solution that the enzyme is in will be too high and contribute to indiscriminate digestion). I always use 10 U of enzyme per µg of plasmid, so if digesting 1 µg, use 10 U (which for example would be 5 µl of a 2000 U/ml enzyme) 5 µl of 10X buffer, 1 µg of plasmid, and up to 50 µl with water. Just to check a plasmid, 1 hr at 37ºC (or whatever temperature the enzyme should be incubated at) is typically enough. For complete digestion for cloning, I will digest for 4 h. Never digest longer than recommended by the manufacturer, as many enzymes will, after a time, begin indiscriminately digesting.
I will hook up with the question, maybe someone could advise.
I have attempted to linearize plasmids with EcoRI and XbaI. Below I upload a electrophoretic separation of 5 plasmid preps (uncut and RE digested).
Can it be assumed (for example) that XbaI-digested plasmid isolated from strain #3 is
a) around ~33kb?
b) and it consists of ~ 23kb and ~10kb fragments after digestion?
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