Transformation of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Investigator: Jeff, Leonie, Rosario

Aim of the experiment: Transformation of Phytochrome B for protein fusion.

Procedure:

• CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

• 2 µl of DNA was added to 100 µl of competent cells and gently mixed.

• 30 min incubation on ice

• 5 min. heat shock at 37 °C

• Adding of 1 ml LB-medium to each tube.

• Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

• 100 µl of the cell suspension was plated on one chloramphenicol plate.

• The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

• The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Picking of of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Investigator: Jeff, Leonie, Rosario, Florian

Aim of the experiment: Picking of of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Procedure:

• pSB1C3 plasmid with BBa_K801031 (PhyB 2 - 908 aa, RFC25): Colonies were picked from chloramphenicol plates.

• Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x).

• 4 colonies were picked.

• These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight