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Oasis Oasis
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11 years ago
I conducted a PCR reaction using a total of 2 ul of 10mM dNTP's.  The product is exactly 100 bp, and 35 cycles were run.  I am now conducting a restriction enzyme digestion using SmaI, which will cut the DNA at only a single site.  Our lab's protocols state simply to allow the digestion to run overnight.  I wanted to know if there was a more defined time needed for a complete reaction, as I'd like to begin an assay with the digestion product as early in the morning as possible.

The digestion was begun at 5:00 pm.  What is the earliest I can safely conclude that the digestion is complete
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wrote...
11 years ago
It's been a while since I did restriction digestion, but if you have good quality enzyme, it should cut within an hour. SmaI digests at 25C, so make sure to get the right temperature (most other enzymes cut best at 37C).
Maybe your lab has a batch of old restriction enzymes, and so they might not work that well anymore.

Other things to remember is that your input DNA should not be more than 10% of your digestion volume, and to get an optimum buffer.

It is probably best to do a second digestion, in a different tube, with a standard plasmid that you know has 1 or 2 SmaI sites, as a control.

Good luck!
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