Engineering BioBrick vectors from BioBrick parts/Colony PCR protocol

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Solutions/reagents:

Parameters:

  • X - concentration of primers (µmole/µl)

Equipment:

  • Thermocycler
  • Sterile 0.6-ml tubes

Steps:

  1. PCR mix
    Use the following table as a checklist for preparing the reaction in sterile 0.6-ml microcentrifuge tube (1):

     PCR SuperMix High FidelityVF2 primerVR primercolony suspensionde-ionized water
    Colony PCR9 µl0.125/X0.125/X1 µlMake up the volume to 20 µl
  2. PCR conditions
    Program a standard thermocycler to run the reaction using the following parameters:
    Initial denaturation
    • Denature: 95°C, 15 mins
    Thermocycling
    • No. of cycles: 39
    • Denature: 94°C, 30 secs
    • Anneal: 62°C, 30 secs
    • Elongate: 68°C, 3.5 mins
    Termination
    • Elongate: 68°C, 20 mins
    • Hold: 4°C, until removed from machine

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 3 hrs, 35 mins

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