I am having issues with my restriction digests and I'm not sure if it is my enzymes, my DNA template or ME! I am setting up a 50ul reaction as follows: 5ul Required buffer, 1ul enzyme, 1ug Template DNA and MilliQ H2O to volume. Reactions are digested a minimum of 1hr. Some of my reactions when run on a gel look great...nice crisp single bands while some(cut with the same enzyme) look like a smeary mess...
What about digestion temperature? How did you stain the gel? If you are seeing nice completely digested bands, but some of them look messy, maybe its a problem with your DNA template... it may be degraded. Have you checked integrity and quality of your DNA samples before doing the digestion?
I spec the DNA before I use it in the reactions...not sure this tells me anything of the quality though. I am running the reactions at the temperatures that are suggested by the enzyme company and also using the recommended buffers. My gel is stained with SYBR safe. The issue is in the DNA i think. I run reactions done with the same enzyme but different templates and they look completely different. One is a nice crisp band and the other not so much...
Which company enzymes you are using and you did double digestion or single digestion ??? Might be the digestion buffer is not compatible check it with company description, take 1ug of DNA for the digestion. Keep reaction volume 50-100 ul at 37 degree for 30-40 minutes. It would be help.
I suggest checking what is recommended for each enzyme. Make sure you are using the recommened buffer, tempreture and time. You could also check the quality and concentration of your DNA template. You mentioned that sometuimes you see smeary mess which makes me think that it has something to do with the prepration of your DNA template. Maybe your DNA pellete had still some of alcohol before you resuspend it in water.
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