Line 489: | Line 489: | ||
<p>Vortex and spin down PCR tube. Put tube on a thermocycler.</p> | <p>Vortex and spin down PCR tube. Put tube on a thermocycler.</p> | ||
− | |||
<table class="table"> | <table class="table"> | ||
<thead> | <thead> | ||
Line 495: | Line 494: | ||
<th>Step</th> | <th>Step</th> | ||
<th>Temperature in °C</th> | <th>Temperature in °C</th> | ||
− | <th>Time in | + | <th>Time in minutes</th> |
</tr> | </tr> | ||
</thead> | </thead> | ||
Line 535: | Line 534: | ||
<p>Golden Gate Mix is ready for transformation. | <p>Golden Gate Mix is ready for transformation. | ||
</p> | </p> | ||
+ | |||
+ | <h4 id="goldengate">Golden Gate Assembly</h4> | ||
+ | <p></p> | ||
+ | <h5>Golden Gate Assembly with BsaI / BpiI</h5> | ||
+ | |||
+ | <p>For 20µL reaction volume pipette in a PCR Tube:</p> | ||
+ | <table class="table"> | ||
+ | <thead> | ||
+ | <tr> | ||
+ | <th>Substance</th> | ||
+ | <th>Amount</th> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>Empty Backbone 40 nM</td> | ||
+ | <td>1 µL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Backbone Insert(s) 40 nM</td> | ||
+ | <td>2 µL each</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>linear DNA Insert(s) 40 nM</td> | ||
+ | <td>4 µL each</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ATP 10x (aliquoted)</td> | ||
+ | <td>2 µL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10x CutSmart Buffer</td> | ||
+ | <td>2 µL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T4 Ligase 10x diluted </td> | ||
+ | <td>1 µL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BpiI/BsaI</td> | ||
+ | <td>1 µL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>AD</td> | ||
+ | <td>variable</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>total volume</td> | ||
+ | <td>20 µL</td> | ||
+ | </tr> | ||
+ | |||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
+ | <p>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. | ||
+ | </p> | ||
+ | |||
+ | <table class="table"> | ||
+ | <thead> | ||
+ | <tr> | ||
+ | <th>Step</th> | ||
+ | <th>Temperature in °C</th> | ||
+ | <th>Time in minutes</th> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>37</td> | ||
+ | <td>2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2 → 1 10x</td> | ||
+ | <td>16</td> | ||
+ | <td>5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>37</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>55</td> | ||
+ | <td>30</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>80</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td>23</td> | ||
+ | <td>10 seconds</td> | ||
+ | </tr> | ||
+ | |||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
+ | <p>Golden Gate Mix is ready for transformation. | ||
+ | </p> | ||
+ | |||
+ | |||
</div> | </div> |
Revision as of 09:11, 7 August 2017
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.