LAB4 - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

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material#LAB4

Summary:

• Wash competent yeast cells
• Preparation of DNA mixture
• Yeast transformation
• Plate transformants on solid SD medium from LAB3

1. Decant the supernatant slowly without disturbing the cells.
2. Pour the content of the yeast culture into a 1.5 mL Eppendorf tube
3. Spin for 20 s in a micro-centrifuge at top speed
4. Remove supernatant by decanting (pipette not needed).
5. Add 1 mL ultra pure water.
6. Resuspend cells by pipetting slowly with a P1000 pipette (slowly, don't make froth).
7. Spin for 20 s in a micro-centrifuge at top speed
8. Remove supernatant by decanting (pipette not needed).
9. Add 800 µL ultra pure water.
10. Resuspend cells by pipetting slowly with a P1000 pipette (slowly!).
11. Put the tube on ice.

We need two mixtures, one complete that we call "➕" and one that lacks the pBR fragment that we call "🔺" (delta)", see Table #5. The ∆ mix is a negative control where we would expect few transformants. The plan is for 12 students to transform with the "➕" mix and 4 students with the "🔺" mix.

1. Pool all successful PCR products together except the tubes with the pBR fragment.
2. Measure the volume using a pipette ( for example 380 µL)
3. Divide the mixture 3/4 (➕) and 1/4 (🔺) (285 µL and 95 µL)
4. Add the pBR fragment to the ➕ mix (70 µL)
5. Add an equal portion of water to the 🔺 mix (70/3 = 23 µL)
6. Add 500 µL water to the ➕ mix (to have enough volume for 12 x 60 µL)
7. Add 167 µL water to the 🔺 mix (to have enough volume for 4 x 60 µL)
8. Mix by vortexing.
9. Divide ➕ mix into 3 tubes, 285 µL in each (one for each group).

Table#5	PCR product	➕ mix (12 student)	🔺 mix (4 students)
	amp	✅	✅
	2µ	✅	✅
	TRP1	✅	✅
	Δcrp	✅	✅
	pBR	✅	❌
	ddH2O	❌	✅

Each student should make one transformation. This protocol is described in detail here.

1. Mix washed cells by inverting the tube.
2. Transfer 67 µL of the cell suspension to a clean 1.5 mL Eppendorf tube per group member (If the group has four members, you need four tubes.). Mark the tubes with your initials.
3. Centrifuge the cells for 20s at the highest speed of the microcentrifuge.
4. Remove supernatant with a P200 pipette. Leave the cell pellet at the bottom of the tube, do not resuspend.
5. Add 40 µL of ➕ or 🔺 DNA mix to the tube with cells.
6. Add 200 µL PLS (PEG-LiAc-ssDNA). Be careful and pipette slowly as PLS is sticky. Use a P1000 pipette with blue tip.
7. Vortex the tubes until cells are well resuspended and no clumps visible.
8. Put the tubes in a floating tube rack at 42°C.
9. Incubate for 40 min.
10. Mark a Petri dish with the appropriate solid medium with your group number and name. Write on the back side of th eplate, not on the lid.
11. Add about 1/2 mL glass spheres (~10-15 spheres) to the Petri dish.
12. Time for ☕ break.
13. Remove tube from water bath after 40 min and put tube on ice for at least 2 min.
14. Spin tube for 20s at highest speed.
15. Remove supernatant with a P200 pipette. Leave the cell pellet at the bottom of the tube.
16. Add 300 µL YPD medium and resuspend with the pipette by slowly pipetting up and down. Be careful as the cells are sensitive
17. Transfer 100 µL of the cell suspension to your Petri dish.
18. Spread the cells by shaking the glass spheres (The samba method).
19. Give the rest of the cell suspension to the instructor
20. Incubate the plates upside down for 2-4 days at 30°C.

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