Lab 3 Procedure

Select Yeast Colonies

• Obtain your plates and identify the yeast colonies.
• Work in a group of 2, circle and number 8 yeast colonies with a marker (fewer if there are fewer colonies).
  • Since the same reaction was used for both plates, choosing any colonies from either or both plates are fine.

• If you have 8 colonies, take out 8 PCR tubes and number them from 1-8. If you have fewer than 8, then use fewer tubes.
• Add 20 μL of 20 mM NaOH into each tube.
• With a 10 μL pipette tip, touch colony #1 slightly.

• Dip the pipette into the NaOH solution in tube #1. Mix well by pipetting up and down. Dispose of the tip.
• Repeat the process with the rest of the colonies.
• Put the PCR tubes in a thermocycler and heat for 10 minutes at 100 °C.
• Place the PCR tubes on ice immediately afterwards.
• Save the yeast plates to be used in the next lab period at 4 °C.

Prepare for Multiplex PCR Reactions:

• Take out a new set of PCR tubes, and label them with numbers 1-8 (or fewer if you have fewer colonies).
• Add 1 μL of yeast DNA into the corresponding new PCR tube.
• Obtain a PCR tube that contains 1 μL of the wild-type yeast DNA (i.e. with no transformation).
• Put side the 9 PCR tubes, each containing 1 μL of the DNA of one colony of yeast cells.
• Set up a master mix for the remaining 19μL of reaction (to make a 20 μL reaction) to be used in all 9 PCR tubes.*
• * A master mix is desirable in this condition because aside from the DNA, the rest of the reagents are the same for all tubes. To minimize pipetting errors, a master mix should be made, mix well and then distributed among the PCR tubes.

      * Prepare 11 reactions if you have 9 PCR samples. If fewer, prepare PCR samples +2 reactions in the master mix. More reactions are prepared to account for the lost of liquid during pipetting/transferring between tubes.

      ** Phusion should be added last.

• Pipette the master mix up and down after adding all the reagents. Avoid introducing bubbles.
• Pipette 19 μL of PCR master mix into each labeled PCR tube. Mix well after each addition. Be sure to change tips after each addition and mixing.
• Once all the components have been added to the tubes, mix them with a pipette or by gently flicking the tubes. Do not vortex!
• Centrifuge the PCR tubes briefly using the small nanotube centrifuge on your bench. You can spin the tiny PCR tubes by placing them inside a 1.5 or 2 mL microfuge tube without the cap (you can break the cap of the tubes).
• Put the PCR tubes in a thermocycler and run the following conditions:
  • Initial denaturation: 95 °C for 3 minutes
  • Denaturation: 95 °C for 15 seconds
  • Annealing: 60 °C for 30 seconds
  • Extension: 72 °C for 1 minute and 15 seconds
  • Repeat steps 2-4 for 35 cycles
  • Final extension: 72 °C for 3 minutes
  • Hold at 10 °C

Figure 3.5 A PCR machine in an open position (left) and in a locked/running position (right)

Make 2% agarose gels

• The following video demonstrates the gel pouring process in the 207 lab. Note that our pouring systems may be different than those you’ve used in other labs:

• Each pod (composed of four people) should prepare one batch of 50 mL agarose and use it to pour two gels.
• Calculate the amount of agarose you will need for 50 mL of a 2% gel.
  • Be mindful that the agarose gel uses a weight-by-volume (w/v) weighting system. This means, for example, to make a 100 mL 1.2% w/v sodium chloride solution, one will need 1.2 gram of sodium chloride and add water up to 100 mL.

• Weight out the agarose and add it to a 250 mL Erlenmeyer flask.
• Add 50 mL of 1 X TAE buffer to the flask.
  • TAE stands for Tris-Acetate-EDTA. A recipe for TAE, and most buffers used in this course, can be found in Appendix 4 for future reference.
• Note: The amount of agarose you will be adding to the TAE is small, so you can use 50 mL of TAE.
• Note: 1 X TAE buffer will also be used as the running buffer for gel electrophoresis in future labs. Therefore, the gel and the running buffer will have the same ionic strength. Failure to use the running buffer to prepare the gel will result in erratic separation of DNA, or no running at all when current is applied.
• Place an inverted 25 mL Erlenmeyer flask in the mouth of the 250 mL flask and heat in a microwave for 50 seconds, swirl to mix, and then heat for additional 10 seconds. If the agarose is not completely melted, repeat the swirl and the 10 seconds.

• Remove the small Erlenmeyer flask and let the agarose solution cool to the touch.
• 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. You can also try to apply a small quantity of water on the clear plasmid gel casting units before sliding; water may ease the sliding friction.
• Once the agarose is cool to the touch, add 2.5 μL of ethidium bromide (EtBr), and gently swirl to mix. Do not remove the EtBr solution from the designated area. Use the designated pipette and dispose of the pipette tip to the appropriate tip beaker.
• Pour approximately half of the agarose into each casting unit (~25 mL each; eyeballing is fine). Immediately add a comb to each gel. Use the 10-well side.

• Wait about 10 minutes 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, use a sheet of Saran wrap to wrap the gel by sliding the gel from the gel holder onto the wrap.
• Write down your section number and name on the Saran wrap. The gels will be used in later labs.
• Place the gels in designated areas.