Caesium chloride gradient Materials and Equipment

• Mistral 3000 centrifuge

• Beckman Optimal TL Ultracentrifuge with TL100 rotor • Bench top microfuge capable of 13,000rpm

• Minigel equipment and power pack

• Chloramphenicol (20pg/ml in Ethanol) (Sigma) • GTE (see appendix 2)

• LB Broth (see appendix 2)

• Sodium Dodecyl Sulphate/Sodium Hydroxide (SDS/NaOH) (see appendix) • Isopropanol

• 3M Sodium Acetate

• TE (10:10) (see appendix 2) • TO.IE (see appendix 2)

• Ethidium Bromide (Sigma) stock solution: lOmg/ml ethidium bromide in TE 10:10

The vector was supplied in DH10B cells by Pieter de Jong (Children’s Hospital Oakland Research Institute, USA) (http://bacpac.chori.org/pbace36.htm).

CsCl gradient purification is still the most efficient way of removing the host

chromosomal DNA from the solution, leaving only the purified vector to be recovered and is described below.

1. E. coli cells containing the vector were spread from an agar stab onto an agar plate containing 20pg/ml chloramphenicol and grown at 37°C overnight. 2. A single colony was inoculated into 200ml of LB broth containing 20pg/ml

chloramphenicol (x4) and grown 37°C overnight.

3. Cultures (4x200ml) were harvested in 250ml tubes and put into a GSA rotor and spun at 5000rpm for lOmins at 4°C.

4. Each pellet was then washed in 10ml of GTE. The suspensions were then combined and re-spun to pellet the cells.

5. The cells were then re-suspended in 10ml GTE and left at room temperature for lOmins.

6. The suspension was split (2x5ml) into Oakridge centrifuge tubes and 10ml of SDS/NaOH was added to each. The tubes were then mixed gently by inverting slowly and left at room temperature for lOmins.

7. Sodium Acetate (7.5ml) was added to each tube. The tubes were mixed and left on ice for 30mins.

8. The cell debris was pelleted by centrifuging the tubes at 10,000rpm for 20mins at 4°C in a SS34 rotor.

9. The supernatants were poured off gently and put into clean tubes. Isopropanol (12ml) was added and left for 15mins at room temperature.

10. The nucleic acid was pelleted by spinning at 15,000rpm for 30mins. 11. The supernatant was decanted off and the pellets washed in 70% ethanol. 12. The ethanol was decanted and the pellets left to air dry.

14. Into two 50 ml Falcon tubes, 4.78g of Caesium Chloride (CsCl) were weighed out. The DNA solution was then added to each tube and the tubes swirled gently to dissolve the CsCl.

15. To each tube, 462pl of ethidium bromide stock solution was added. 16. The tubes were spun at 3000rpm at RT for 12mins to pellet the debris.

17. A 16G needle was attached to a 5ml syringe. The supernatant was recovered using the syringe and the contents emptied into a 5.1ml Beckman ultracentrifuge tube. The meniscus had to reach the base of the spout of the tube.

18. A metal sealer was placed on the top of the tube and a heated Beckman adaptor tool was placed on the top of this. With pressure and heat, the tube would seal by melting down the collar of the neck. Once the neck was sealed, a cooling tool would be placed on top of the metal sealer for a few seconds. All tools should be either provided with the centrifuge or the tubes.

19. The tubes were then places into a rotor with spacers and the rotor was placed into an ultracentrifuge.

20. The tubes were then spun at 70,000rpm for 24 hours at 20°C.

21. When the spin was finished, the tubes were carefully removed from the rotor and placed in a rack.

22. The tubes were then illuminated with long wavelength U/V light. This would show up the bands that contained the vector and the cellular debris. Figure 5 describes how the vector was removed:

Figure 5.

The tube was pierced with a needle here

A 20G needle was fitted to a syringe inserted into the tube just

below the broad vector band. The

flat edge at the end of the needle

Cellular debris

23. The removed vector was then placed back into a fresh, clean Beckman tube and the liquid topped up with stock CsCl and ethidium bromide/TE solution and the tubes sealed again.

24. The tubes were then spun overnight at 20°C and 70,000rpm.

25. The DNA was removed as before, but then split between two eppendorf tubes. There should be approximately 2x600pl.

26. An equal volume of isobutanol was added to each tube. The tubes were then vortexed and then spun in a microfuge for lmin at 13,000rpm. This was repeated by multiple isobutanol extraction until both layers colourless.

27. The lower layer containing the vector DNA was removed with a pipette and placed into a dialysis bag.

28. The bag was dialysed against 250ml TO.IE at 4°C, changing the TE every hour 5 times.

29. The vector DNA was recovered from the bag and placed into an eppendorf tube. 30. Added to this was 1/10 volume of sodium acetate then 2 volumes of ethanol. 31. This was then incubated overnight at -20°C.

32. The tube was then spun at 13,000rpm in a microfuge for 30mins to pellet the DNA. The supernatant was removed and the pellet allowed to dry.

33. The DNA was then resuspended in 120pi TO. IE.

34. A small amount of this was then run on a agarose minigel (0.8%) to check the yield.

2.2.1.2 Stuffer fragment removal