Lab 6 Procedure
Select Yeast Colonies
- Obtain your plates and identify the yeast colonies.
- Work in a group of 4, 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.
Note: Only write on the outside of the plate!
- 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.
Note: You only need a tiny bit of the colony. Too much DNA will negatively affect the PCR result.
- 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)
- Set up a master mix for 20 μL reactions:
| Reagent | Stock Conditions | Final Conditions | 1 Reaction (RXN) | Master mix of 11 RXN* | |
| Reaction Buffer | 5X | 1X | |||
| dNTP | 10mM | 200 μM | |||
| Primer mix | 10 μM | 400nM | |||
| H2O | |||||
| Phusion** | 1U/ μL | 1U | |||
| Total Volume | 19 μL | 209 μL |
* 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.
Please note that the reaction volume is 20 μL and not 50 μL as was used in lab 2. If you are not sure about the calculation, please consult with the teaching team.
- 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.
- 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
Make 2% agarose gels
- Working in a group of 4, make two 2% agarose gels. Refer back to Lab 2 for detailed instruction. Note that the gels are 2% instead of the 1% gels that were made in lab 2.
- The 2% gels will be used in the next lab.
Note: EtBr is a carcinogen (cancer-causing)! When working with it, please observe the following precautions:
- Work with the provided EtBr stock only in the designated area.
- Use the designated EtBr pipette. Dispose of pipette tips in the designated EtBr tip waste container.
- Wear gloves and eye protection.
- Dispose of EtBr gels in the designated containers.