Biology Protocols

• Set up a starter culture in 10-15ml LB broth containing the required antibiotic. Incubate overnight at 37°C.
• Inoculate 4ml of the starter culture into 2 x 200ml LB broth containing the required antibiotic.
• Incubate the cultures at 37°C with shaking (220rpm) for 16hrs.
  

ALL WORK TO BE DONE ON ICE

• Harvest the cells by centrifugation (5K; 10m) and resuspend the cells in 3ml of lysis buffer (25% sucrose in 0.05M Tris.HCl pH8.0 and 25mM EDTA).
• Add 0.5ml of 5mg/ml lysozyme in 0.05M Tris.HCl pH8.0.
• Mix and incubate on ice for 2 minutes (timing is critical here, if the cells look like lysing, i.e., go glassy, continue with stage 10.
• Add 4ml Brij-Doc buffer:

8g Brij58
3.2g sodium deoxycholate
in 860ml water
then add 40ml Tris.HCl pH8.0
and 100ml EDTA pH8.0

• Mix by inverting the tube slowly. Incubate on ice for no more than 2 minutes. The solution should become opalescent but not too viscous.
• Centrifuge (10K; 30m; 4°C).
• The pellet should be small and compact. Remove 7ml of the supernatant (containing the plasmid) to a plastic universal bottle.
• Add 1g CsCl per ml of supernatant (therefore 7g). Dissolve the CsCl and then add 250µl ethidium bromide (10mg/ml).
• Put the supernatant in a Beckman "bomb" centrifuge tube. Make up the volume and balance with paraffin oil and then seal the tube.
• Place in a 70.1Ti rotor and centrifuge overnight (50K; 16-20h; R/T).
• Remove the tubes from the centrifuge and examine under UV light. The tubes should contain two bands, the lower one being the plasmid.
• Remove the lower band by puncturing the top of the tube with a 16G syringe needle and then puncturing the tube again just below the plasmid band. Slowly remove the band into a syringe.
• Put the plasmid band in a small plastic tube with a cap and remove the ethidium bromide as follows:
• Add an equal volume of CsCl-saturated isopropanol and shake gently. The ethidium bromide will enter the alcoholic phase. Remove the isopropanol and repeat at least 3 times until the aqueous phase is colourless.
• Transfer the aqueous layer to a 15ml Corex tube and add 2 volumes of TE buffer. Then add 2.5 volumes of absolute alcohol and mix.
• Place the tube in a -20°C freezer overnight.
• Centrifuge the solution (10K; 30m; 4°C).
• Remove the alcohol by tipping it off. Dry the DNA pellet. Resuspend the pellet in 500µl of TE buffer. Transfer the DNA to a 1.5ml eppendorf tube.
• Check for the presence of the plasmid by agarose gel electrophoresis.