DNA extraction kit Macherey & Nagel. Extraction was performed according to protocol. The leaf pieces were either frozen with liquid nitrogen and afterwards added to the mortar and pestle or water was added at the this step.
Open the Pollen:
Ribolyser:
6500 rpms*2 two times performed*30 seconds duration of the rotation*30 seconds pause; Hersteller?
Media (cultivating bacteria):
LB = 20g LB powder filled up to 1L
Competent cells KRX from promega were used for transformation. Heat shock transformation were tested with the promega provided protocol and with the protocol from the iGEM Bielefeld team 2014:
Transformation via heat shock
Thaw 200 μL Chemo competent E.coli cells on ice
Add 0.5-5μL plasmid to 200 μL chemocompetent cells
Store cells on ice for 10-30 minutes
Heatshock for (60-)90 seconds at 42°C
Transfer transformation reaction to 1 mL SOC or LB Medium and incubate 1 h at 37°C
Centrifuge 30 seconds at 11000 rpm and plate on selective LB Medium
Incubate overnight at 37°C
Plasmid isolation was performed with the analytic Jena plasmid isolation kit. The enclosed protocol was used. It was changed a bit as the elution step was done with water instead of elution buffer.
PCR:
Plant PCR were performed with Synadorme mastermix with the following protocol:
Phusion PCR for cloning was performed with fermentas Phusion enzyme and the following protocol:
- 5x Phusion HF buffer (4µl)
- 10mM dNTPs (0,4µl)
- Forward primer (1µl)
- Reverse Primer (1µl)
- Template DNA (1µl>
- Phusion DNA polymerase (0,2µl)
- H2O (add to 20µl)
Gel electrophoresis:
TAE, 50x stock solution (provided by the supervisors); working solution 1x TAE
0,8%-2% agarose (welche?) dissolved in 1x TAE
50x TAE:
242 g Tris Base
57.1 mL Glacial Acetic Acid
100 mL 0.5 M EDTA
pH:7.8
Nanodrop 2000 from XY was used for DNA measurements.
Restrictionsenzyme?
The experiments with liquid nitrogen and trypsin as well as the primer design strategy were especially supported by the supervisors. No antibiotics were handled by team members.