Volume problem - (Nov/27/2008 )

When ever i put my restriction digestion setup on the water bath and after the 65 degree treatment to kill the enzyme. My volume of the mixture is always low as compared to the original volume. Does its usual in some body experiment or not. Or may be the our water bath is showing the wrong temperature.
Thanks.

Liquid tend to evaporate easily in tubes. Try to centrifuge your tubes after they cooled down.

If you get smaller volume after that, then your tubes are leaking, and that's not a good news.

Hi, i use to add loading dye with xylen cyanol, bromphenol blue EDTA ant SDS to kill enzyme. If you plan to separate fragments on agarose gell, then simply load it and run and excise from gel
good luck

Can you give me the recepie for the loading buffer you used to kill the enzyme and how much time you kept it and at which temperature (room temperature I hope).

regards

If I am gel purifying a digest I just run the digested DNA with no inactivation. The gel purification protocol is enough to kill an trace amounts of enzyme that might be left over.

If I do need to heat-inactivate an enzyme at 65°C, I just make sure the tube is tightly closed, and then spin it down afterwards to collect any evaporated water that has condensed on the tube lid.

Easy peasy.

Ginger