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 1min, and then centrifuge.

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 mins 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.