Molecular Cloning Tips

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Molecular Cloning Tips

Make sure digestions are totally digested
1. It is easier to cut from a vector that has an insert than one that doesn’t
2. As controls digest vector with one enzyme only (if one of the enzyme doesn’t cut, that lane will run perhaps slightly lower as a supercoiled form?)
3. For a double digest, digest first with the less efficient enzyme to prevent the generation of a oligonucleotide overhang where the less efficient enzyme won’t cut well
4. Then add in the more efficient enzyme (make sure that it can cut the overhang in two hours or so).
5. Perhaps purify before using alkaline intestinal phosphatase (for 30uL, use 1 mL in NEBuffer 2 for 1 hour)
6. Run on a gel at 80V for as long as possible, and use long-wave UV (preparative) to cut it out before taking a photo of it (careful to not cut out any contaminating bands
7. Digestion of vector should be at about 2 hours, and longer if required
8. For PCRs, run on a gel, cut out correct band, then purify before doing any digestions; digestions should be done overnight if required
9. During the ligation step, calculate the correct ratio of insert:vector; in one of the samples, use 3x as much insert
10. If during winter, room temperature ligation may not really be at high enough temperature to do for only 2 hours, so it may be required to do it for longer (e.g. 5 hours?)