Hi,
Please take a look at the restriction digest blot I have attached here. I ran the reaction twice with changing the parameters a little but still the same result! Here's what I did:
Enzymes used: Nhe1 and SacII. Four lanes: lane1: double digest with Nhe1 and SacII, lane2: Only Nhe1, Lane3: SacII, Lane4: Uncut. I am expecting to see two bands: 6400bp and 5700bp. But you can see there is no change between all the four lanes. I did troubleshooting after the first trial and made some changes for the second reaction but I got the same result.
Here's the composition of my reaction mixture: total 50 uL
DNA - 0.84 uL(1ug)
Nhe1- 1uL (10 units)
sacII- 0.5uL (10 units)
10X NEB 2.1 buffer - 5uL
6X EZ vision DNA dye- 8uL
Deionized H2O - 34.66 uL
This composition is for the double digest mixture and for single cuts I omitted one of the enzymes and made up the volume to 50uL. These enzymes can cut within 15 minutes but in my second trial I incubated them at 37 degrees for 30 minutes to ensure complete digest. Used 0.7% agarose gel and ran it at 4v/cm for about 2 hours. I am trying to figure out why all the lanes are showing bands of the same size when they shouldn't. I am suspecting if it is DNA methylation that is restricting the enzymes used from cutting the DNA but if so the uncut lane should be different from the rest, which I don't see. the feeling I have now is my DNA sample is not good enough and I might have to redo the Maxiprep of the plasmid. If anybody has any suggestion or clue about this please let me know. I appreciate it.
Thanks
Why are you adding the 6X EZ Vision DNA dye to the restriction digest? I suspect the dye is binding to the DNA and preventing the restriction enzymes from cuttting the DNA. Have you tried digesting without the DNA dye then adding it after the digest is complete and running the gel?
Why are you adding the 6X EZ Vision DNA dye to the restriction digest? I suspect the dye is binding to the DNA and preventing the restriction enzymes from cuttting the DNA. Have you tried digesting without the DNA dye then adding it after the digest is complete and running the gel?
i agree with Tom that ez dye does bind to dna and you should cut in its absence. One other possibility is that this is the wrong template and it does not have the 2 enzyme cut sites
Add the dye after the incubation, also if I remind well both these enzymes are Star activity-free so you could extend easily your digestion up to 1h.
Try again and let us know, good luck!
I also agree with all the above answers and suggestions, but I would first check for two things; first, please check if you have the correct DNA samples and if its the correct DNA samples then check for contamination with other DNA fragments. second, please check your buffers and enzymes (may be they were forgetting on the bench) and also the water, could any of these be contaminated with other DNA or the salt concentrations in the buffer or in the water. so it could be one of these two reasons or combinations of both, for example the DNA is contaminated and the buffer or the enzymes not working as it should be.
you could also try to digest different plasmid as a positive control to test the enzymes and the reaction components and the Dye. Please Let us know and GOOD LUCK
Thanks for all the suggestions. I use the EZ dye to get away with using Ethidium bromide. . I have been adding the dye before digestion but I can see how EZ dye can interfere with digestion and it makes sense to add the dye after the digestion step. I will go ahead and try that today and see how it turns out. As far as the state of enzymes and buffers go I am confident that they are fine as they are new. I used a common buffer which allows 100% activity for both the enzymes and I use deionized water. Therefore, I think I am fine as far as the quality of reaction mixture goes. I will post what I find today after I add the dye post digestion. Thanks a lot again for all the suggestions.
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