yeast transformation short - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

This practical class consists of three separate tasks.

1. Prepare a DNA mixture.
2. Yeast transformation using the DNA mix
3. Assemble the final plasmid on your computer.

Each student should prepare a mixture of the PCR products obtained in the previous class. The teacher has pooled all the successful PCR reaction in their respective categories:

1. ampR
2. pBR
3. ΔCRP
4. CEN_ARS / 2µ
5. LEU2 / TRP1 / HIS3 / TRP1

The specific PCR reactions used can be found in the "DNA_pool" tab of the Google sheet. There is one 1.5 mL Eppendorf tube with each component. There is **only one** tube of each kind, so share the tubes in a practical way between groups.

Each student should prepare the DNA mixture outlined in the "TRAFO_plan" tab of the Google sheet. Note that some students prepare a mix with five components (we call this the "+" mix) and some with four components and water (we call this the ∆ mix). The delta (∆) mix has all the components except the pBR fragment.

1. Calculate how much is needed of the + and ∆ mixes (count the number of students with each mix)
2. Each student need 40 µL of mixture = 5 components * 8 µL
3. Prepare the mixture in a fresh Eppendorf tube and mix well.
4. Prepare one fresh Eppendorf tube for each student.
5. Mark these tubes with the "TRAFO#" numbers. These numbers can be read from the Google sheet, tab "TRAFO_plan"
6. Divide 40 µL mix into the correct tubes with the correct numbers.

This is the only material available, so be sure to pipette the correct volumes and do not contaminate the tubes as other students will use them.

Each student should make one transformation.

1. Take two new 1.5 mL Eppendorf tubes.

2. Transfer 1 mL **ultra pure water** to one tube and put it on ice. Cold water is needed later in the protocol.

3. Transfer 50 µL of the cell suspension to the other tube. Mark this tube with your "TRAFO#" number.

4. Centrifuge the cells for 20s at the highest speed.

5. Remove supernatant with a P200 pipette. Leave the cell pellet at the bottom of the tube, do **not** resuspend.

6. Add 30 µL of your **DNA mix** (+ or ∆ )to the tube with cells.

7. Add 150 µL PLS (PEG-LiAc-ssDNA) Be careful and pipette slowly as PLS is sticky. Using a P1000 pipette might be easier than using a P200.

8. Vortex the tubes until the cells are well resuspended.

9. Put the tubes in a floating tube rack at 42°C.

10. Incubate for 40 min.

11. During this time you should do the DNA assembly on your computer.

12. Mark a Petri dish with the appropriate solid medium with your "TRAFO#" number and name. Write on the back side, not on the lid.

13. Add about 1/2 mL glass spheres (~10-15 spheres) to one empty Petri dishes with solid medium.

14. Remove tube from water bath after 40 min and put on ice for at least 2 min.

15. Spin tube for 20s at highest speed.

16. Remove supernatant with a P200 pipette. Leave the cell pellet at the bottom of the tube.

17. Add 100 µL cold water (from the tube on ice, step 1) and resuspend with the pipette by **slowly** pipetting up and down.

18. Transfer all of the cell suspension to yout Petri dish.

19. Spread the cells by shaking the glass spheres (The samba method).

20. Incubate the plates upside down for 2-3 days at 30°C.