SMALL Scale Transformation

 

SMALL Scale Transformation

 

 

1. Inoculate 1 ml of YPDA or SD with several 2�3 mm colonies.

2. Vortex vigorously to disperse any clumps.

3. Transfer cells to a flask containing 50 ml of YPDA or SD:

4. Incubate at 30 o C for 16�18 hr with shaking (250 rpm) to stationary phase (OD600>1.5).

5. Transfer overnight culture (enough to produce an OD600 = 0.2�0.3) into 300 ml of YPDA

6. Incubate at 30 o C for 3 hr with shaking (230�270 rpm).

7. Place cells in 50-ml tubes and centrifuge at 1,000 x g for 5 min at room temperature.

8. Discard the supernatant and resuspend cell pellets by vortexing in 25�50 ml of sterile TE or H2O

9. Pool cells centrifuge at 1,000 x g for 5 min at room temperature.

10. Decant the supernatant.

11. Resuspend the cell pellet in 1.5 ml of freshly prepared, sterile 1X TE/LiAc:

12. Prepare 10 ml PEG/LiAc solution.

13. In the indicated tube, mix the following a :

• DNA-BD/bait 0.1 µ g

• AD/library 0.1 µ g

• Herring testes carrier DNA 0.1 mg (10 µ l)

14. Add 0.1 ml of yeast competent cells and mix well by vortexing.

15. Add 0.6 ml of sterile PEG/LiAc solution and vortex at high speed to mix.

16. Incubate at 30 o C for 30 min with shaking (200 rpm).

17. Add 70 µ l of DMSO. Mix well by gentle inversion or swirling. Do not vortex.

18. Heat shock for 15 min in a 42 o C water bath.

19. Chill cells on ice for 1�2 min.

20. Centrifuge cells for 5 sec at room temperature at 14 K rpm

21. Remove the supernatant.

22. Resuspend cells in 0.5 ml of 1X TE b :

23. Proceed to plating (on either SD �Trp/�Leu) or SD �Trp/�Leu/�His/�Ade with or without X- á -gal as required.