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 1ml LB-medium to each tube.

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

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