Difficult double digestion - (May/07/2001 )
I have a MCS in a vector. The restriction enzymes are EagI and SfiI. It is easy to cut the vector in a single digest but a double digest is difficult to detect as the fragment that is removed is only 14bp and is difficult to see the differnece on a gel. Is there any other way of detecting this digest
If you are cloning, just try to clone your insert in, after preping the colonies you'll easily be able to see those which contain insert and those which dont.Or cut the vector with both enzymes indiviually to ensure both are working properly!
If any of the two enzymes cuts well in the exact conditions of double digestion, you can be almost sure that everything is all right.So, if you are afraid that somthing might be wrong, make two controlswithout one of the enzymes in parallel with your restriction.
Do a control ligation with the double digested vector and follow by a control transformation