I am currently using Amicon Ultra centrifuge tubes that are built to concentrate DNA. Unfortunately, I seem to be loosing almost 90% of my sample between running the sucrose gradient and concentrating. How are samples normally dealt with after a sucrose gradient?
Note: I have been ending up with ~12ml of sucrose+DNA after running the gradient, so I need a way to handle larger volumes.
Sucrose has fairly low solubility in ethanol. I worry that the sucrose will drop out with the DNA, and then I'm stuck with a bunch of sucrose in my sample.
Another option is dialysis. Be sure to include EDTA in the sample and dialysis bath to inhibit DNase activity. After the dialysis removes the sucrose, you can employ DNA precipitation.
do u really need sucrose gradient? I think the problem comes from sucrose gradient not Amicon Ultra centrifuge, I am using them daily with regular and modified DNA with mw more than 4000 and they working fine
Thank you everyone for the input. The problem was too much DNA in the Amicon tubes. It was precipitating out and sticking to the membrane. I was able to modify a few things to make the tubes work for me.
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