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Why loading buffer can't go down after enzyme digestion?

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#1 lilin_hust



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Posted 05 February 2014 - 09:29 PM

I digest the plasmids with Roche enzyme SacI, and use buffer A. After inactivation, I add loading buffer, but the loading buffer can't precipitate? How is that?


But if I just use the original not digested plasmids, the loading buffer won't float. So it seems the floating is related to enzyme digestion?


Does anyone ever met this case?? Thanks so much for those who can give me some tips!!

#2 perneseblue


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Posted 06 February 2014 - 06:13 PM

Just to make sure we are on the same page, do you mean that the digest and loading buffer will not sink to the bottom of the well?


If so, may I ask if you ethanol percipitated your digest before adding the loading buffer?


If ethanol percipitation was used, what you have is alcohol contamination in your tube when the loading buffer was added. You need to remove the alcohol by drying it.

If you are willing to accept slightly ugiler DNA bands and willing to run the gel electrophoresis tank a little slower, you can add the loading buffer directly to the digest (no inactivation) and run it straight onto the gel.

May your PCR products be long, your protocols short and your boss on holiday

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