Cell Fractionation ProtocolCell Fractionation Protocol

Make sure the digestive really digest Cell Fractionation Protocol

1. It is easier to cut from the vector that has an insert of one who doesnt
2. As a control vector digested with an enzyme only (if one of the cutting enzyme does, that the line will run probably slightly lower as supercoiled form?)
3. To double digest, digesting the first with less efficient enzyme to prevent the generation of oligonucleotide overhang where less efficient enzyme wont cut well
4. Then add in a more efficient enzyme (make sure that it can cut the overhang in two hours or more).
5. It may purify before using intestinal alkaline phosphatase (for 30uL, use 1 mL in NEBuffer 2 for 1 hour)
6. Run the gel at 80V for as long as possible, and using UV wavelengths (preparative) to stop it before taking a photo of it (being careful not to cut any contaminating band.

Make sure the digestive really digest Cell Fractionation Protocol

7. Digestion of the vector should be around 2 hours, and longer if necessary
8. For the PCR run on the gel, cut out the band properly, then purify before making any digestion; digestions be done overnight if necessary
9. During the ligation step, calculate the correct ratio of insert: vector; in one sample, using 3x as much as the insert
10. If, during the winter, the room temperature ligation may not actually be at a temperature high enough to do only two hours, so it may be necessary to take longer (eg 5 hours?)