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. Pelet 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 frrom 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 clarifed 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 Bufer CW to ISLOATE 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.