Engineering BioBrick vectors from BioBrick parts/Colony PCR

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Contents

Materials

  • PCR SuperMix High Fidelity
  • VF2 primer (5'-TGCCACCTGACGTCTAAGAA-3')
  • VR primer (5'-ATTACCGCCTTTGAGTGAGC-3')
  • Deionized, sterile H2O
  • strip of PCR tubes
  • 2-log DNA ladder
  • 0.8% E-Gel® from Invitrogen Corporation in Carlsbad, CA

Equipment

  • DNA Engine OPTICON\texttrademark from MJ Research, Inc. (now Bio-Rad Laboratories, Inc., Hercules, CA)
  • E-Gel® PowerBase™ v.4 from Invitrogen Corporation in Carlsbad, CA
  • Alpha Innotech FluoChem™ 880 gel imaging system

Procedure

PCR mix

  • 9 μL PCR SuperMix High Fidelity
  • 6.25 pmoles VF2 primer
  • 6.25 pmoles VR primer
  • 1 μL colony suspension
    • dilute 1 colony in 100 μL water

PCR conditions

  1. 95°C for 15 minutes
  2. 94°C for 30 seconds
  3. 62°C for 30 seconds
  4. 68°C for 3.5 minutes
  5. Repeat 2-4 39 times.
  6. 68°C for 20 mins
  7. 4°C forever

Agarose gel electrophoresis

  1. Dilute the reactions four-fold with water.
  2. Perform an agarose gel electrophoresis of 20 μL of each diluted reaction using a 0.8% E-Gel®.
  3. Also electrophorese 1 μg of 2-log DNA ladder to verify the length of each PCR product.
  4. Image gel with 302 nm transilluminating ultraviolet light using an ethidium bromide camera filter and an exposure time of 614 milliseconds.
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