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.
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.
