Problem
You will purify three 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 µL 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.
• Place the QIAQUICK spin column into a new labelled 1.5 mL microcentrifuge tube.
• To elute the DNA add 30 µL 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 prepare a: 1/50 dilution in a final volume of 200 µL.
Task
• Why do we perform a second centrifugation after adding the wash buffer?
• What volumes of DNA and of water will you need for this dilution?