My double digested plasmid and insert are almost the same size and it is impossible to cut the insert from the gel as it falls behind the plasmid even on a 2% gel.
I have been thinking of using a third or fourth enzyme to cut the background plasmid further so that I can separate the insert. I prefer using a FastDigest or HF enzymes but I have never gone beyond double digestion. Will it work?
I still haven't decided what third or fourth enzyme to buy, so I wanted to learn this before purchase.
Yes, triple or quadruple digestions work, as long as the buffer is compatible with all three or four enzymes, and as long as their recognition sites are not too close to each other (as a rule of thumb, they should be separated by at least 4 bp -though you won't have that problem in your scenario).
And another rule of thumb: Try to keep the combined volume of all enzymes under 10% of total reaction volume. The problem is that most enzymes are stored in buffers containing 50% glycerol, and glycerol relaxes recognition specificity for many enzymes at concentrations higher than 5%.
Hope it helps!
Yes, adding third enzyme targeting the vector would be the easiest way and it works well. But you need to check that that enzyme is not is your insert and the buffer is compatible with all enzymes as well.
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