Hi all,
I am extracting natural plasmids from my bacteria. I don't know the size, nor the sequence. I am trying to determine plasmid profiles of the bacterial isolates I have. I have bands on the gel after the extraction, that should correspond to the plasmids, Since the size of them looks big, I am trying to digest them with restriction enzymes in order to cut them in fragments that can be measureable once I run them on the gel. After trying different enzymes, I was not able to see any fragment on the gel after the digestion..I tried to run also the uncut DNA (what I have prepared is a sort of negative control, mixing all the components of the digestion mix except for the enzyme) but I was not able to see this either. I thought that the lack of detection on the gel could be due to the diluition of the DNA when I set the digestion mix. I add 3 ul in 21 ul of water. Indeed, when I try to diluite my DNA in water (without setting up any restrction enzyme digestion) i am not able to see anything on the gel. So, i was wondering if it is likely that I don't see any product because the DNA is too diluited to be seen on the gel. Is it possible?What do you suggest? Can you suggest me something?
Thanks
It sounds as though you could be having a dilution problem or something else going on. We first have to see how far you are diluting your plasmids and how much of the dilution you are loading on the gel.
When you add your DNA to your digestion mix (- or + enzyme), how much of the diluted DNA are you loading on your gel? If you are running 3 ul of the uncut, are you running the entire reaction containing 3 ul of the plasmid sample or just a fraction of that, like just 3 ul? If it is just 3 ul of a 20 or 21 ul digestion, then you are loading only 1/7th the amount of DNA as your uncut.
If I were running 3 ul uncut to see the bands(s) clearly, I would set up the digestion with more DNA in it, so when you run a portion of your digestion on the gel, you are still loading at least the equivalent of 3 ul of uncut plasmid on the gel. If you are using 4-cutter enzymes, you could be cutting your plasmid into such a high number of fragments that none of them is above your limit of detection. So you may need to play around with how much of the digestion to overload onto the gel to see the smaller fragments.
You might also be having trouble with diluting the DNA in water before loading onto the gel. I would recommend using a buffered solution such as TE or 10 mM Tris-HCl, pH 8.
Try to be very specific with describing your protocol with volumes of each component, which enzymes, how much was loaded on the gel, and perhaps we will be able to find any problems more easily.
Since you say that simply diluting the plasmid in water without any digestion makes it no longer visible, it does seem like you are indeed diluting too much/loading too little dna on your gel. This effect is further enhanced if your enzyme cuts the plasmid into several bands, since each band will have an even lower concentration, as pointed out by Christopher S Morello.
I agree with the answer above.
You can also esitmate the concentration of DNA in your plasmid prep by spectrophotometry etc. Then use about 0.5microgram DNA in 25microL volume reaction for RE digest. Try to cut with 6 or 8 cutter enzymes which would result in bigger fragments compared to digesting with 4 cutter enzymes.
thank you all for your answers.what do you suggest to visialize my dna better?can I reduice yhe volume of water
I routinely perform 10 ul digests so that can contain 8 ul of DNA and the rest being enzyme and buffer. Then I can add 2 ul of 6X dye and add the entire digest into the well if the starting DNA concentration was low.
You only have to be careful not to add more than 1 ul of RE to the reaction tube or the high glycerol concentration from the enzyme may either inhibit/slow the reaction or promote star activity. Best is making an enzyme master mix with the enzyme and the buffer so that 1) you can use 1 ul of enzyme (which is probably 10 U and 1 or 2 U should be plenty for ≤ 1 ug of plasmid DNA) which both prevents star activity and high glycerol concentration AND also allows you to add as much volume of DNA in the digest as possible, and 2) ensures that each reaction gets the same enzyme and buffer amounts.
Since we still don't know how much of your undiluted DNA you can visualize, what percentage of that you are loading from your digestion, or any other specifics, I would suggest loading 1X and/or 2X the DNA mass on the gel relative to the uncut. If the volume of uncut DNA you are loading leads to a faint (nearing the limit of detection), then increase the amount of all DNAs on the gel.
Once your sure that you've loaded a mass of DNA from the digestion that is the same or 2X higher than the uncut plasmid which you can visualize on that gel, if you still can't see your digested DNA then you have some reagent problem. Try diluting your ladder in the same water/buffer and then loading the whole volume on the gel to see if the ladder is affected. If you try using some 6 and 8 cutter REs like Sonam Mehrotra suggests, then you might find an enzyme that cuts the plasmid 0 or 1 times and you should definitely see those.
Lastly, you asked about reducing the volume of water for the DNA. For the digests, that's answered above. For the uncut plasmid, you only need to dilute your DNA enough to contain the loading dye and to be at a volume that just covers the bottom of the well: usually around 1 ul per mm of gel well width for standard thickness wells. You'll know if the volume was too little if the resulting band(s) are not the full width of the well or if the bands are not continuous from end-to-end.
Good luck.
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