IGEM:British Columbia/Protocols/3A Assembly
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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
- Perform a restriction digest on all the parts you wish to assemble:
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.