Lab 3: PROCEDURE

Agarose Gel Electrophoresis

Each pod (composed of four people) will prepare one batch of 50 mL agarose and use it to pour two gels. Two people will then share one gel to load their samples.

• Calculate the amount of agarose you will need for 50 mL of a 1% gel. Weigh out the agarose and add it to a 250 mL Erlenmeyer flask. Next, add 50 mL 1× TAE buffer to the flask. (TAE stands for “Tris–Acetate–EDTA.” A recipe for TAE, and for most buffers used in this course, can be found in the Appendix for future reference: Appendix 4: Buffer formulations)

The following video demonstrates the gel pouring process in 551 lab. Note that our pouring systems may be different than those you’ve used in other labs.

• Place an inverted 25 mL Erlenmeyer flask or wadded up paper towel in the mouth of the 250 mL flask and heat in a microwave until all the agarose has completely dissolved. In our microwave, try heating the gel for 45 seconds, swirl to ensure thorough mixing of the agarose, and then heat for additional 15 second intervals until the solution is clear.

• Allow the transparent agarose solution to cool until you can hold the flask in your hand. You should start restriction digestion while waiting for the gel to cool down (see below).
• Meanwhile, place the black rubber bumpers onto the ends of the clear plastic gel casting units. The bumpers are a tight fit so they may be difficult to put onto the casting unit. If you have difficulty sliding the bumpers onto the casting unit, try sliding one side on and then the other. Once you have the bumpers on, place the comb into the unit.
• Once the agarose is cool, add 2.5 μL EtBr (10 mg/mL), and gently swirl to mix. Do not remove the EtBr solution from the designated area. Use the designated pipette.
• Fill each casting unit with half the agarose (~25 mL each; eyeballing is fine).
• Immediately add a comb into the each gel. The side of the comb used depends on the number of samples you plan to run.

• Wait about 5–10 min for the gel to solidify; the gel should appear cloudy, not clear.
• Once the gel has solidified, gently pull the comb straight up out of the gel. Be careful not to destroy the wells when you pull out the comb! Next, remove the bumpers by gently twisting them off. After removing the comb and the bumpers, place the casting unit into the running box. Both casting units from your pod will fit into the same running box.

• Add approximately 350 mL of 1× TAE buffer to the running unit so that the buffer just covers the gel. Make sure that both gels are covered with buffer. If there is used TAE buffer available, please use that.
• For each of your PCR reactions, place 5 μL H2O into a new microfuge tube, then add 5 of your PCR reaction. Be sure you pipette the PCR reaction all the way down into the water in the tube. Add 2 μL of 6× loading dye and mix by pipetting up and down.
• Load 5 μL of the 1 kb ladder onto the gel. Load all 12 μL of your PCR+loading dye samples onto the gel in separate wells.

• Once all of the samples have been loaded into both gels, gently place the cover on the running box (be careful not to disrupt your samples). Attach leads to power supply. Set the voltage at 150 V and press the run button.

• Continue electrophoresis until the bromphenol blue (the leading, darker dye) has migrated to about 1/2 of the way to the end of the gel.
• Take a picture of your gel using the gel doc, with help from the teaching staff.
• Please pour your TAE buffer into the “Used TAE” carboy so it can be re-used.

Restriction Digest

Set aside a sample of each PCR reaction to run on your gel, and begin digesting the remaining DNA as soon as you microwave your agarose (before making the gel is fine as well)!

You will be digesting all three PCR products (the HCAII gene and the pETblue2 vector and the negative control) with DpnI. Fortunately for us, DpnI is functional in the PCR reaction buffer. If the enzyme was not compatible with this buffer, you would have to purify the DNA from the reaction, and then digest.

• Add 1 μL DpnI directly to each PCR tube. You should digest all three reactions – including the negative control
• Mix the reactions well by gently flicking the tube with your finger.

• Briefly centrifuge the samples and incubate at 37 °C for 45 minutes.

DNA Purification Column

• After you have run your gel and determined which reactions contain the desired PCR products, and completed the DpnI digests, you will purify the DNA using small spin columns. Note you are also purifying your negative control. Use one column for each reaction. Be sure to label the spin columns so you don’t get them mixed up!

• Add 5× volume of PB buffer to your PCR reactions. (Example: if you have 100 μL of sample, add 500 μL PB). Mix well before moving on to step 3.
• Add the PB buffer + PCR reaction solution (from step 2), to the spin column and spin for 1 min. at 14,000 rpm (>16,000 rcf) in the centrifuge. Be sure to balance the tubes in the centrifuge before you start spinning. Your DNA should now be bound to the column.
• Add 750 μL Buffer PE and spin for 1 min. at 14,000 rpm.
• Spin for 1 minute at 14,000 rpm again to dry the column. (Buffer PE contains ethanol, which will interfere with the Gibson assembly reactions if it is not completely removed).
• Place the column into a clean 1.5 mL eppendorf tube labeled with the name of the sample, the date, and your initials.
• Add 30 μL of Elution buffer (EB; 10 mM Tris pH 8.5) to the center of the column and incubate for 1 minute at room temperature.
• Spin for 1 minute at 14,000 rpm in the centrifuge. The flow-through contains your purified product. In order to get the maximum amount of DNA off of the column, add the flow-through back to the center of the column, incubate for one additional minute and then spin for 1 min. at 14,000 rpm in the centrifuge.
• SAVE THE FLOW-THROUGH CONTAINING YOUR DNA.

Determining the DNA Concentration

Once you have purified your PCR product, you will need to determine the concentration of your product. You will use the NanoDrop spectrophotometer to do this.

The following video demonstrates use of the NanoDrop:

• Place 2 μL of EB on the platform of the nanodrop and click on “BLANK” (upper left-hand corner) on the computer.
• Once the blank has been completed, place 2 μL of your purified PCR product onto the platform and click on “MEASURE”. Write down the DNA concentration and the 260/280 and 260/230 ratios.

Preparing for Next Lab

You should place your purified DNA samples into the class freezer box for storage at -20 until next week.

Clean up

• Empty tip jar to the “USED TIPS” box (note that the box is for used tips only; all other non-sharps, such as gloves, should go to regular trash).
• Dispose of or put back reagents (see blackboard for details).
• Dispose of used columns, PCR tubes, eppendorf tubes in regular trash.
• Dispose of used gels in gel container.
• Pour used gel running buffer back to the carboy, and wash gel running cassette.
• Put everything from the drawers back to the corresponding drawers. If supplies (such as beakers) are used to hold liquid, wash the supplies and leave them on the bench to dry. Do NOT dry them on the drying rack above the sink.
• SAVE purified DNA, EB, 1kb ladder and 6x gel loading dye in the common freezer box.