IGEM:British Columbia/Protocols/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.
 * Perform a restriction digest on all the parts you wish to assemble: