______—Module 4 –

PURPOSE: To extract recombinant plasmid from putatively transformed HB101.

MATERIALS:

PROCEDURE:

1.  Prep:

2.  Distribute all 10 ml of saturated the cell culture to 1.5 ml µfuge tubes.

3.  Spin @ top speed in a µfuge for 2m.

4.  Discard super, tap on paper towels to get rid of any residual liquid.

5.  Resuspend the entire sample in 100 µl TEG (Tris-EDTA-Glucose) buffer. After resuspending, you should have all 10 mls of the original overnight culture resuspended in 100 µl TGE.

6.  Mix by pipetting (as you combine portions) and vortexing.

7.  Add 0.2ml of fresh NaOH/SDS cell lysis solution.

8.  Gently mix by inverting and tapping.

9.  Incubate on ice for 5 minutes.

10. Add 0.15ml of potassium acetate solution. Gently mix by inverting and tapping.

11. Incubate on ice for 5 minutes.

12. Centrifuge in a microcentrifuge (14,000 rpm) for 5 minutes. Make sure the centrifuge is balanced.

13. Remove 0.4ml of the supernatant to a fresh 1.5ml µfuge tube. Try to avoid drawing in the white precipitate. Set the old tube aside. Initial or put your lab group number on the tube.

14. Thoroughly mix the deproteinization matrix (N) and quickly add 0.4ml to your sample.

15. Mix the sample with matrix thoroughly by vortexing and inverting for 3 minutes.

16. Centrifuge 5 minutes in a microcentrifuge. Make sure the centrifuge is balanced.

17. Transfer 0.4ml of the aqueous phase (upper) to a fresh 1.5ml µfuge tube. Initial or put your lab group number on the tube. Discard the old tube.

18. Add 0.8ml of ice-cold 95-100% ethanol. Mix well by inversion.

19. Incubate the tube on ice for 20 minutes.

20. Place your tube (with balance) in the microcentrifuge so that the tab which connects the lid to the tube is facing the outside of the rotor. Centrifuge for 10 minutes at 14,000 rpm (top speed).

21. Remove and discard all the supernatant with a Pasteur pipette or automatic pipette. Be careful not to dislodge the pellet which will be on the lower wall of the tube or at the bottom. Do not touch the inner wall of the tube that was facing the outside of the rotor.

22. Carefully add 0.25ml of ice cold 80% ethanol. Keep a close eye on the pellet while doing this since it may dislodge at this step. Vortex, spin.

23. Immediately but carefully remove all the ethanol with a pipette, take care not to aspirate the pellet.

24. Alternatively, carefully pour out the ethanol. This step rinses the tube of residual salts.

25. Air dry the tube 20 minutes or place under vacuum for 5 minutes.

26. Add 50µl TER (Tris-EDTA-RNase) buffer. Resuspend by thoroughly vortexing.