Difference between revisions of "Team:BOKU-Vienna/Protocol"

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     <ul>
 
     <ul>
 
         <li class="small"><a href="#transformation">Transformation of chemically competent E. Coli</a></li>
 
         <li class="small"><a href="#transformation">Transformation of chemically competent E. Coli</a></li>
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    <ul>
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        <li class="small"><a href="#transformationyeast">Preparation of electro-competent Pichia pastoris cells</a></li>
 
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<p> 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.</p>
 
<p> 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.</p>
  
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<h4 id="transformationyeast">Preparation of electro-competent Pichia pastoris cells</h4>
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<p></p>
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<p>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,
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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.
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</p>
  
 
<h4 id="miniprep">Miniprep</h4>
 
<h4 id="miniprep">Miniprep</h4>

Revision as of 12:13, 7 August 2017

Protocol

V

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.