IGEM:British Columbia/Protocols/3A Assembly

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Contents

3A Assembly

Materials

  • Construction Plasmid
  • Prefix biobrick part
  • Suffix biobrick part
  • Absolute Ethanol (100% = 200 proof)(Ethanol Precipitation) or PCR purificaion kit
  • 95% ethanol (Ethanol Precipitation) or PCR purification kit
  • T4 DNA Ligase
  • Ligase buffer (1uL aliquots)
  • sdH2O
  • Competent Cells
  • LB agar plates (with antibiotic)
  • Agarose (1g/100mL for a 1% gel)
  • 0.5X TBE Buffer (dilute 10X TBE into diH2O)
  • Loading buffer
  • SYBRSafe
  • 1/20 dilutions of 100bp or 1kb ladder

Equipment

  • Tabletop centrifuge
  • -80°C freezer
  • Vortex
  • Water bath (at 16°C and 42°C)
  • Ice
  • Gel box, combs and casting tray
  • Miniprep kit

Method

Construction plasmid (EcoRI, PstI), Prefix part (EcoRI, XbaI), Suffix Part (SpeI, PstI). 2. Purify restriction digests: Use Ethanol precipitation for parts that are <200bp, or a Qiagen PCR Purification Kit for parts >200bp. 3. Ligate all parts together. 4. Transform 10μL of ligation reaction into DH5α competent cells. 5. Perform Colony PCR on colonies to confirm insert length/presence. 6. Streak out correct colony on LB Agar plate with the correct antibiotic, inoculate overnight culture, miniprep and store.

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