Hi, everyone.
I have many problems when doing the DNA fragmentation assay (agrose gel eletrophoresis). 1. The genome DNA was isolated through the ethanol method, but it don't dissolve in the TE buffer. 2. The solution is too viscous to be loaded in the agarose gel. 3. How about the sampling amount of the DNA? according to cell numbers or DNA quatification （The DNA solution is not well disolved to quatify it accurately ?
Hope someone could help me. Thanks very much
Hi Shirley,
What is your TE buffer concentration? Try 10mM Tris and 1mM EDTA buffer solution to dissolve the DNA. If the solution still viscous you can add little more Nuclease free water to dilute. If you not need to store the DNA for long time, you can use Nuclease free water/ autoclaved water instead of TE buffer. I think it may work.
thank you and best wishes.
Shirley, sometimes along with the DNA, additional compounds could been extracted, e.g. polyphenolics, some carbohydrates, etc. so such compounds should be blamed for the high viscosity of a solution, not DNA. DNA should dissolve well in TE and even in water overnight. If not, try to optimize your extraction procedure to get rid of contaminating compounds.
Shirley-
Q1. R U extracting DNA from tissues or isolated cells from cell culture?
Q2. Viscosity means high DNA concentration (dense milky white color).
If you answer these questions, I may be able to provide some suggestions.
SDR
Thank you all.
Answer for Sidhartha's question:
Q1: I extract DNA from cultured cancer cells (lysis buffer-NaAc-ethanol )
Q2: After add TE buffer to the extracted DNA, the solution is not only viscous, but also with some insoluble precipitation (cotton-like) in it.
Mostly according to this protocol: 3*106 cells were collected and in the end only 30ul TE buffer were used to dissolve it. I suppose the reason maybe there are some additional impurities, but also be afraid of lose small fragments of DNA. So would you suggest me a proper protocol ? Thanks very much!
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