Revision as of 09:54, 17 June 2010 by Swoodhouse (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

PCR purification protocol

We used the QIAquick PCR Purification bench protocol.

1. Add 5 volumes of Buffer PBI to 1 volume of the PCR reaction and mix. If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
2. Place a QIAquick column in a 2 ml collection tube.
3. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30-60s. Discard flow-through and place the column back into the same tube.
4. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30-60s. Discard flow-through and place the column back into the same tube.
5. Centrifuge the column in a 2 ml collection tube for 1 min.
6. Place each column in a clean 1.5 ml microcentrifuge tube.
7. To elute DNA, add 50 µl water ti the center of the QIAquick membrame and centrifuge the column for 1 min. For increased DNA concentration, add 30 ml elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.

DNA ligation protocol

1. Add ...
2. Add 1µl ligation buffer to the tube.
3. Add appropriate amount of insert to the tube
4. Add ..
5. Add 0.5µl ligase.

Transformation protocol

1. Thaw a 200µl aliquot of the desired strain of E. coli and add the transforming DNA (10 ng of vector DNA in 10 µl). Incubate for 45 min at 42°C.
2. Heat-shock the cells for 120 secs, and place on ice again for 3-4 min.
3. Add 1ml of LB broth and incubate the cells at 37°C for 1-15 hr in a water bath with gentle shaking.
4. Plate out 100-200µl/plate on LB (agar at 1.5%), containing the appropriate selection markers.
5. Incubate plates overnight at 37°C.