Hi everyone, I'm currently working with a plasmid called pBI101, I isolated this plasmid from a plate then I did the extraction with mini prep protocol and cut it with SmaI at 25°C incubation temperature. After that I visualized the digestion in agarose gel 1% but coudn't see anything, meanwhile the control (plasmid without cutting) was there. Why am I losing it? I'd be glad if someone could help me, I'm using NEB enzymes for the digest.
I think that Sma1 usually cuts at 37c so possibly cutting at 25c Allows star activity and the many small fragments generated are too faint to see
Agreed, SmaI does cut at 37. Do you set up your uncut plasmid exactly the same as the cut (with the omission of the enzyme of course)? Meaning, the same amount of DNA, in a tube, with the buffer and water, and in the incubator? Be sure to cut at 37.
You could also try shorter incubation times (15 min, 1 hour) to see if you are getting the predicted product size vs. many fragments.
We always used SmaI at 25oC so there should not be an issue with star activity but it will work at 30 or 37 (just less stable if I recall). I think the control that Amy mentions is important, you may have some nuclease contamination in your plasmid prep, so when you restriction buffer you might be activating the nuclease.
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