Team:BOKU-Vienna/Protocol
Protocol
Protocols from A-Z.
Our protocols will follow shortly.
Contents
Materials
LB medium - E. Coli
| Name | Amount |
|---|---|
| Hy Soy Peptone | 10.0g |
| Yeast Extract | 5.0g |
| NaCl | 5.0g |
| RO-water | 1000 mL |
LB Agar - E. Coli
| Name | Amount |
|---|---|
| Hy soy Peptone | 10.0g |
| Yeast Extract | 5.0g |
| NaCl | 5.0g |
| RO-water | 980 mL |
| Agar-Agar per 500mL | 7g |
Methods
Transformation of chemically competent E. Coli
Competent cells from E. Coli strain DH5α or DH10B were thawed on ice. The whole ligation/other DNA samples were added to the cells, for 30 min the mix was put on ice. The cells and DNA mix were heat shocked for 90s at 42°C. The cells recovered on ice for 5min. 1 mL of LB medium was added to the cells and then an incubation followed at 37°C in a shaker-incubator for 45min. An appropiate amount of cells were plated on a selective LB agar.
Preparation of electro-competent Pichia pastoris cells
Pichia pastoris cells are generated, which are competent for transformation by electroporation. For the pre-culture, inoculate a selective YPD medium and incubate over night on a shaker. For the main culture, inoculate non-selective YPD medium with the pre-culture and incubate again over night. On the next day, measure the OD and split the culture into two falcon tubes. Centrifuge the cells and discard the supernatant. Add pre-treating solution to each of the pellets, resuspend and incubate the cells for 30 minutes. Add ice-cold sorbitol to both Falcon-tubes and harvest cells, then discard the supernatant. Combine the pellets of the two tubes and resuspend in ice-cold sorbitol. Harvest them and discard the supernatant. Resuspend the cells in sorbitol and store it in Eppendorf tubes on ice until transformation. For electoporation, an aliquot of the cells is mixed with linearized DNA. As negative control, cells are transformed with elution solution or AD. Transfer the mix into a cooled electroporation cuvette and incubate it on ice for 5 minutes. Set the parameters on the electroporator to 2000 V, 25µF and 186 Ω. After transformation 1 mL of YPD-media is added to the cells in the cuvette, then they are transferred into a microcentrifuge tube. Regenerate the cells for 1.5h-3h at 28°C. Then plate some of the cells onto selective agar plates and incubate at 28°C until colonies appear. The rest is kept on agar plates at 4°C.
Miniprep
Minipreps were performed using Hi Yield®Plasmid Mini DNA Isolationkit according to the manufacturer's protocols.
InterLab Study
The Interlab Challenge was performed according to the protocols for the InterLab Study in 2017. For further details visit the iGEM homepage: InterLab Study or our InterLab page.
Kit Plate 6 InterLab Part Locations
| Name | Backbone |
|---|---|
| Positive Control | BBa_I20270 |
| Negative Control | BBa_R0040 |
| Test Device 1 | BBa_J364000 |
| Test Device 2 | BBa_J364001 |
| Test Device 3 | BBa_J364002 |
| Test Device 4 | BBa_J364003 |
| Test Device 5 | BBa_J364004 |
| Test Device 6 | BBa_J364005 |
Q5 PCR
| Name | Amount |
|---|---|
| in PCR tube aliquoted oneTaq 2x MM | 12.5 µL |
| AD | variable |
| Primer forward | 1.25 µL |
| Primer reverse | 1.25 µL |
| Template | 1 ng |
| Total Volume | 25 µL |
Vortex and spin down PCR tube. Put tube on a thermocycler.
PCR programme: NebQ5
| Step | Temperature in °C | Time in seconds |
|---|---|---|
| 1 | 98 | 30 |
| 2 | 98 | 10 |
| 3 | variable - Primer annealing temperature | 30 |
| 4 → 2 30x | 72 | variable - 1 min/kb |
| 5 | 72 | 3 min |
| 6 | 23 | 10 |
Add 5 µL of 6x Loading Dye for preparative gel electrophoresis.
Golden Gate Assembly
Golden Gate Assembly with BsaI
For 20µL reaction volume pipette in a PCR Tube:
| Substance | Amount |
|---|---|
| Empty Backbone 40 nM | 1 µL |
| Backbone Insert(s) 40 nM | 2 µL each |
| linear DNA Insert(s) 40 nM | 4 µL each |
| GG Mix 20x | 1 µL |
| GG Buffer 10x | 2 µL |
| AD | variable |
| total volume | 20 µL |
Vortex and spin down PCR tube. Put tube on a thermocycler.
| Step | Temperature in °C | Time in minutes |
|---|---|---|
| 1 | 37 | 2 |
| 2 → 1 10x | 16 | 5 |
| 3 | 37 | 10 |
| 4 | 55 | 30 |
| 5 | 80 | 10 |
| 6 | 23 | 10 |
Golden Gate Mix is ready for transformation.
Golden Gate Assembly
Golden Gate Assembly with BsaI / BpiI
For 20µL reaction volume pipette in a PCR Tube:
| Substance | Amount |
|---|---|
| Empty Backbone 40 nM | 1 µL |
| Backbone Insert(s) 40 nM | 2 µL each |
| linear DNA Insert(s) 40 nM | 4 µL each |
| ATP 10x (aliquoted) | 2 µL |
| 10x CutSmart Buffer | 2 µL |
| T4 Ligase 10x diluted | 1 µL |
| BpiI/BsaI | 1 µL |
| AD | variable |
| total volume | 20 µL |
T4 Ligase has to be diluted 1:10 (0.5 µL T4 and 4.5 µL AD). Vortex and spin down PCR tube. Put tube on a thermocycler.
| Step | Temperature in °C | Time in minutes |
|---|---|---|
| 1 | 37 | 2 |
| 2 → 1 10x | 16 | 5 |
| 3 | 37 | 10 |
| 4 | 55 | 30 |
| 5 | 80 | 10 |
| 6 | 23 | 10 seconds |
Golden Gate Mix is ready for transformation.
OneTaq Colony PCR
| Substance | Amount |
|---|---|
| in PCR tube aliquoted oneTaq 2x MM | 6 µL |
| AD | 5 µL |
| Primer forward | 0.5 µL |
| Primer reverse | 0.5 µL |
| Colony | 1 tip |
| Total Volume | 12 µL |
Vortex and spin down PCR tube. Put tube on a thermocycler. PCR programme: OneTaq
| Step | Temperature in °C | Time in seconds |
|---|---|---|
| 1 | 94 | 3 min |
| 2 | 94 | 30 |
| 3 | variable - Primer annealing temperature | 40 |
| 4 → 2 30x | 68 | variable - 30 sec/kb |
| 5 | 68 | 5 min |
PCR Mix is ready for electrophoresis. No loading dye is needed.
