Lab 3 Procedure

Agarose Gel Imaging and Analysis

• Working in a group of 2, take out the one of the two gels you prepared last time.
• Put the gel back onto the gel holder/mold.

• Place the mold into the running box; two molds can fit into one running box.
• Add approximately 350 mL of 1x TAE buffer to the running box so that the buffer just covers the gel. If there is used TAE buffer available, please use that.
• For each of your PCR reactions, place 5 μL H2O to the very bottom of a new microfuge tube, then add 5 of your PCR reaction to the bottom of the tube.
• Add 2 μL of the 6x leading dye and mix the solution gently by pipetting up and down. Try to avoid introducing bubbles.
• Load 10 μL of your dyed PCR reactions and 3 μL of the 1kb ladder into the gel in separate wells.
  • The recommended order for gel 1 is: F1, F2, negative control 1, F3, F4, negative control 2, ladder.
  • The recommended order for gel 2 is: F5, P1, negative control 1, P2, P3, negative control 2, ladder.
  • You can choose other orders, but make sure to write down any order you decide to load on your lab notebook.
• Once all the samples have been loaded into the gels, gently place the cover on the running box. Be sure to match the color between the cover and the electrodes (black with black, red with red).
• Attach leads to the power supply in a color-matched manner.
• Set the voltage at 150 volts (V) and press the run button.

• Continue electrophoresis until the running front (darker dye) has migrated to 1 /2 or 2/3 of the gel.
• Take a picture of your gel using the gel doc, with help from the teaching team.
• Compare the results with the predicted length of each PCR reaction in Appendix I.
• Pour your TAE buffer into the “Used TAE” carboy so it can be reused.

DNA Purification

• After your gel starts running, you can purify the remaining 45 μL of the PCR products using a spin column by following the steps below. Use one column for each reaction.
• Step 1: Add 5x volume of PB buffer to your PCR reactions
  • For example, if you have 50 μL of the sample, add 250 μL PB (hint: since the PCR tubes hold up to 200 μL of liquid. To add 5x volume of PB, you should first transfer the PCR reactions to new 1.5mL microfuge tubes).
  • Mix well.
  • Label the spin columns.
• Step 2: Add the PB/PCR reaction solution to the spin column and spin at 14,000rpm for 1 minute in the tabletop centrifuge.

• • Your DNA should be bound to the column after the spin.
  • Dispose of the flow-through solution.
• Step 3: Add 700 μL PE buffer and spin at 14,000rpm for 1 minute and dispose of the flow-through solution.
• Step 4: Put the empty column back into the centrifuge and spin for 1 minute at 14,000rpm. This step ensures the removal of all solutions.
• Step 5: Add 30 μL of elution buffer (EB) to the center of the column and incubate for 1 minute at room temperature.
  • Remove the top portion of the column (that contains that filter) and put it into a new, labeled microfuge tube.
  • Place the column/tube into the centrifuge.
  • You will not be able to cap the tube due to the column. Place the column/tube in a way that the cap of the tube is the closest to the center of the centrifuge.
  • Spin for 1 minute at 14,000rpm in the centrifuge.
• Step 6: To maximize yield, pipette the 30 μL solution back to the column (that still contains a small amount of your PCR product) and re-spin for 1 minute at 14,000rpm.
• Step 7: Store the purified DNA and the EB in your freezer box.