Lysogeny Broth

Materials

• 10g of tryptone
• 5g of yeast extract
• 10g of NaCl
• 15g of agar
• 1L of Deionised Water

Procedure

1. Use a container a container with at least double the volume of the LA that you are making.
2. Measure out the weights of tryptone, yeast extract, sodium chloride and agar with deionised water to 1l and mix well.
3. Ensure the lid is unscrewed by two and a half turns.
4. Send to be autoclaved.
5. Pour the plates next to a Bunsen burner.
6. Leave for 15-20 minutes to set/solidify.

Mini-Prep or Plasmid Purification

• Harvest bacterial cells
  1. Pellet 20ml of saturated E. coli for 60 seconds at 11,000 x g.
  2. Discard supernatant and remove as much liquid as possible.
• Lyse cells
  1. Add 500ml Resuspension Buffer P1 and resuspend cell pellet by vortexing.
  2. Split the solution into two 1.5ml microcentrifuge tubes.
  3. Add 250μl Lysis Buffer 2.
  4. Mix gently by inverting tube 8 times.
  5. Incubate at room temperature for five minutes or until lysate appears clear.
  6. Add 300μl Neutralization Buffer 3.
  7. Mix thoroughly by inverting tube 8 times.
• Clarification of lysate
  1. Centrifuge for five minutes at 11,000 x g at room temperature
  2. Put 500μl of Buffer PW1 per 1.5ml microcentrifuge tube used in heat block heated to 50օC
• Bind DNA
  1. Place ISOLATE II Plasmid Mini Spin Column in a 2ml Collection Tube
  2. Pipette a maximum of 750μl of clarified sample supernatant onto column
  3. Incubate at room temperature for two minutes.
  4. Centrifuge for one minute at 11,000 x g and discard flow-through.
  5. Repeat stage 4 using the same ISOLATE II Plasmid Mini Spin Column and 2ml Collection Tube with the clarified sample supernatant from the other 1.5ml microcentrifuge tube from the same sample.
• Wash silica membrane
  1. Add 500μl Wash Buffer Pw1
  2. Centrifuge for one minute at 11,000 x g
  3. Add 600μl Wash Buffer PW2 (supplemented with ethanol)
  4. Centrifuge for one minute at 11,000 x g
  5. Discard flow-through and reuse Collection Tube
• Dry silica membrane
  1. Centrifuge for two minutes at 11,000 x g, to remove residual ethanol
  2. Place ISOLATE II Plasmid Mini Spin Column in a 1.5ml microcentrifuge tube.
• Elute DNA
  1. Add 50μl Elution Buffer P directly on the top of the silicon matrix
  2. Incubate at room temperature for two minutes
  3. Centrifuge for one minute at 11,000 x g.

Agarose Gel Extraction

1. Excise and dissolve gel slice
2. Using a clean scalpel excise DNA fragment from gel
3. Remove excess agarose, determine weight of gel slice and transfer into a clean tube
4. Add 200μl Binding Buffer CB per 100mg of 2% agarose gel
5. Incubate sample at 50օC for ten minutes, vortexing sample briefly every three minutes until gel slice is completely dissolved
6. Incubate at room temperature for two minutes
Bind DNA
1. Place ISOLATE II PCR and Gel Column in a 2ml Collection Tube and load 600μl of the sample
2. Centrifuge for thirty seconds at 11,000 x g and discard flow-through
3. Reuse collection tube for step 3
Wash silica membrane
1. Add 700μl Wash Buffer CW to ISOLATE II PCR and Gel Column
2. Centrifuge for thirty seconds at 11,000 x g
3. Discard flow-through and place column back into collection tube
4. Repeat step three to minimize chaotropic salt carry-over
Dry silica membrane
1. Centrifuge for one minute at 11,000 x g, to remove residual ethanol
2. Place ISOLATE II PCR and Gel Column in a 1.5ml microcentrifuge tube

Elute DNA

1. Incubate at room temperature for three minutes
2. Add 15-30μl Elution Buffer C directly onto silica membrane
3. Incubate at room temperature for three minutes
4. Centrifuge for one minute at 11,000 x g.

SOC Media

Materials

To make 100ml SOC Media

• 2g Tryptone
• 0.5g Yeast Extract
• 2.5ml 400mM NaCl
• 625μl 400mM KCl
• 10ml 100mM MgCl2
• 1ml 200mM Autoclaved and filter sterilised Glucose

Procedure

1. Weigh out tryptone and yeast extract into vessel suitable for autoclaving. Add the NaCl, KCl, MgCl2 to the bottle.
2. Make up to 100ml with Distilled Water.
3. Make glucose solution in a vessel suitable for autoclaving.
4. Autoclave both solutions separately to avoid the reaction of glucose with other components.
5. Add 1ml glucose solution using a filter sterilisation syringe to the media.

Competent Cell Production

Materials

• 100ml LB + 5ml for overnight culture
• 100mM CaCl2
• 85mM CaCl2, 15% glycerol v/v

Procedure

1. Streak competent cells onto agar plate and incubate overnight at 37 OC
2. Prepare and autoclave above solutions.
   Inoculate a single colony into 5ml LB in a 50 ml falcon tube. Grow overnight at 37 OC, shaking at 200rpm.
   Keep solutions at 4 OC overnight, and LB at 37 OC so that when cells get transferred they do not experience a temperature change.
3. Pre-cool the rotor of the centrifuge.
   Use 1 ml of overnight culture to inoculate 100ml of LB in a 250ml bottle. Shake at 37 OC for 1.5-3 hours, until OD650 reaches 0.4-0.6.
   Put cells on ice for 10 mins (keep cold from now on, and cool everything on ice before adding). Split into 2 x 50ml falcon tubes.
   Centrifuge in the big centrifuge for 3 mins at 5000 rpm.
   Decant supernatant and gently resuspend in 5ml cold 100mM CaCl2 by inverting tube slowly. (Cells susceptible to mechanical disruption)
   Incubate on ice for 20mins
   Centrifuge as before (3 mins at 5000 rpm)
   Discard supernatant and resuspend in 2.5ml cold 100mM CaCl2/ 15% glycerol v/v
   Pipette into microtubes and freeze in -80OC. (100µl per tube).