Protocol

Protocols from A-Z.

Our protocols will follow shortly.

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.

Team:BOKU-Vienna/Protocol