Making Linearized Plasmid Backbones


Creating Linearized Plasmid Backbone

  • PCR mix
  • Enough water to fill up to 50 uL (23.1 uL)
  • 25 uL of PCR Master Mix
  • 0.7 uL of SB-prep-3P-1 (suffix primer)
  • 0.7 uL of SB-prep-2Ea (prefix primer)
  • 0.5 uL template DNA at 10 ng/ul (5 ng of DNA)
    • Notes:
    • Do not use a sample of linearized plasmid backbones (PCRed) as a template
    • The Registry uses BBa_J04450 as a template
    • Often 5 ng of DNA is too little to measure. We just used 0.5 uL regardless of DNA concentration and our PCR products turned out fine.

PCR Program

  1. 94C/2min
  2. 94C/30s
  3. 55C/30s
  4. 68C/3min
  5. Repeat cycle (steps 2 to 4, 35 more times)
  6. 68C/10min
  7. Digest with DpnI enzyme: 2ul in 100ul reaction, incubate 37C/hour; heat kill 80C/20min

PCR Cleanup

QIAquick PCR Purification

  1. Apply sample to a spin column and add 500 ul Qiagen buffer PB.
  2. Spin through a column twice and centrifuge at 13000 rpm for 60 seconds.
  3. Discard flow through.
  4. Wash 1x with 700 ul buffer PB and centrifuge at 13000 rpm for 60 seconds.
  5. Discard flow through.
  6. Wash 2x with 760 ul buffer PE and centrifuge at 13000 rpm for 60 seconds. Discard flow through each time.
  7. Dry spin at 13000 rpm for 3 min.
  8. Place column in a clean 1.5 mL microcentrifuge tube. Add 50 uL of nuclease free water to the center of the membrane.
  9. Let sit at room temperature for 10 minutes.
  10. Centrifuge the column at 13,000 rpm for one minute to elute.
  11. Repeat steps 8-10 again in the same microcentrifuge tube. You should have 100 uL total when finished.