CsCl Plasmid Prep

Schedule:

• Day -1: Grow cells on a selection plate overnight
• Day 0: Grow single colonies of cells in liquid, under selction overnight
• Day 1: Prepare ultracentrifuge tubes; spin overnight
• Day 2: Isolate DNA from CsCl solution; preciptate overnight
• Day 3: Wash, dry, and resuspend plasmid DNA

Protocol:

1. Use a sterile toothpick to streak frozen cells onto a 2X YT + Amp agar plate. Incubate at 37° overnight.
2. Autoclave 50 ml 2X YT in a 250 ml flask. Make at least 2 flasks.
3. Add 50 to 100 µl of 50 mg/ml Amp to each flask. Pick a single colony of cells into each flask of media; incubate at 37°, with shaking, overnight.
4. Transfer each culture to an Oakridge tube and spin at 8K rpm for 10 minutes in the Sorval with SS-34 rotor.
5. Resuspend each pellet of cells in 4ml TEG using a Pasteur pipette

   [Stock]	Reagent	Amount	[Final]
   1 M	Tris (pH = 8)	2.0 ml	25 mM
   0.5 M	EDTA	0.8 ml	10 mM
   2 M	Glucose	0.8 ml	50 mM
   -	ddH2O	36.7 ml	-
   Total		40 ml

6. Let sit 5 min, at room temperature.
7. Add 8ml fresh lysis solution to each tube; invert to mix; let sit 10 min, on ice.

   [Stock]	Reagent	Amount	[Final]
   10 N	NaOH	1.0 ml	0.2N
   20 %	SDS	2.5 ml	1 %
   -	H2O	46.5 ml	-
   Total:		50 ml

8. Add 6ml ice-cold 3M K 5M Acetate; invert; vortex; let sit 10 min, on ice.

   Reagent	Amount
   KAc	29.44 g
   Glacial Acetic Acid	11.5 ml
   H2O	to 100 ml

9. Spin 10K rpm, 15 min, 4° in the SS34 rotor.
10. Transfer supernatant to sterile 125ml flask; add at least 2 volumes of cold 95% EtOH; let stand at least 10 min at room temperature.
11. Spin half of the solution in a sterile 30ml Corex tube, 8K, 10 min in the SS34 rotor; pour off the supernatant; repeat with the remaining half.
12. Dry the pellet in the Corex tube in the Speedvac (cover with parafilm; poke holes in the film; lay tube in Speedvac under the rotor)
13. Resuspend the dry pellet in TE

    TLA100.3	VTI65
    2.5 ml	4 ml

14. Add CsCl to resuspended plasmid, allow it to dissolve

    TLA100.3	VTI65
    2.5 g	4.21 g

15. Add 10mg/ml EtBr to each Quick Seal tube

    1/2" x 1 1/4"	1/2" x 2"
    200 µl	270 µl

16. Transfer CsCl solution to tube with a Pasteur pipet; balance (paired tubes must not differ by more than 0.1 g); seal.
17. Spin at 22°, for at least 8 hours

    TLA100.3	VTI65
    75k rpm	45k rpm

18. Pull of bottom band (closed circle DNA) with a 1ml syringe and an 18 gauge needle.
19. Extract at least 4 times with equal volumes of NaCl saturated isopropanol (until the isopropanol is clear).
20. Divide into 200µl or less aliquots in microfuge tubes; add 3 volumes of H₂O, 0.06 new volumes of 7.5M NH₄OAc, 0.7 new volumes of isopropanol.
21. Vortex, put at 4° for at least 1 hr.
22. Spin 15 min at 4°, wash with 70% EtOH, dry in the Speedvac.
23. Resuspend in 0.4 ml H20, combining samples that had been divided.
24. Add 0.1 ml 7.5M NH₄OAc and 1 ml 95% EtOH.
25. Leave at -20° overnight.
26. Spin 15 min at 4°.
27. Wash with 70% EtOH, dry in the Speedvac.
28. Resuspend in TE.
29. Check OD₂₆₀
    OD₂₆₀ * 50 * dilution * 10^-3 = µg/µl