Team:UC San Diego/Protocols
- Set up the reactions on ice based on the calculator :
- Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
- Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μl of the assembly reaction, following the transformation protocol.
- Thaw tube of DH5-alpha cells on ice for 10 minutes.
- Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
- Place the mixture on ice for 30 minutes. Do not mix.
- Heat shock at exactly 42°C for exactly 45 seconds. Do not mix.
- Place on ice for 5 minutes. Do not mix.
- Pipette 120 µl of room temperature LB broth into the mixture.
- Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
- Warm selection plates to 37°C.
- Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
- Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours
Component | Volume | Final Concentration |
---|---|---|
10X Buffer for KOD Hot Start DNA Polymerase | 5 ul | 1X |
25 mM MgSO4 | 3 ul | 1.5 mM |
dNTPs(2mM each) | 5 ul | 0.2 mM |
PCR Grade Water | X ul | |
Sense (5') Primer (10 uM) | 1.5 ul | 0.3 uM |
Auto-Sense (3') Primer (10 uM) | 1.5 uL | 0.3 uM |
Template DNA | Y ul | |
KOD Hot Start DNA Polymerase (1 U/ul) | 1 ul | 0.021 U/ul |
Total Reaction Volume | 50 ul |
Component | Volume | Final Concentration |
---|---|---|