You will purify three (3) samples simultaneously in three separate columns: digested GFP amplicon, digested phosphatase treated pUC19, and the digested non-phosphatase treated pUC19.

Add 5 volumes of buffer PB to 1 volume of your reaction and mix by inversion.

Apply the sample to the QIAQUICK spin column and centrifuge for 1 minute.

Discard the flow-through to liquid waste and place the QIAQUICK spin column back into the same tube.

To wash, add 750 L of buffer PE to the column and centrifuge for 1 minute.

Discard the flow-through to liquid waste. Place the QIAQUICK spin column back into the same tube. Centrifuge for 1 minute.

Question: Why do we perform a second centrifugation after adding the wash buffer?

6. Place the QIAQUICK spin column into a new labelled 1.5 mL microcentrifuge tube.

To elute the DNA add 30 L of sterile water to the center of the QIAQUICK spin column, wait 1 minute and then centrifuge for 1 minute.

Measure the concentration of each the three (3) DNA samples that you purified. To measure the concentration you will need to prepare a 1/50 dilution in a final volume of 200 L.

Question: What volumes of DNA and of water will you need for this dilution?

Setup a reaction mixture of 50L to perform the PstI digestion of the GFP amplicon