Team:DTU-Denmark-2/Team/Protocols

Amplifying of biobricks by PCR

PCR MIX

PCR Programs

Purification of PCR Product

Gel electrophoresis

USER cloning

Tranformation in E.coli

Purification of plasmids

Fungi

Transformation in fungi

Mammalian cells

Cell culture and reagents

Medium

• 500 ml DMEM
• 50 ml Fetal Calf Serum (FCS)
• 5 ml Penicillin
• 5 ml Streptomycin

Cultivation of the cells

• Place the appropriate amount of EDTA-trypsin and complete DMEM in the incubator (37°C), before handling the cells (about 1-2 hours before).
• The HeLA and the U2OS cells are kept at -80°C until defrosted gently at 37°.
• Exactly when defrosted, they are mixed with 12 ml complete DMEM in the pipette.
• The cell suspension is transferred to a 15 ml vial and centrifuged at 280 g for 10 minutes. The supernatant is discarded.
• The cell pellet is resuspended in 5 ml complete DMEM and transferred to a 25 cm2 culture flask.
• The cells are kept at 37°C in a 5% CO2 incubator until the following day, where they are passed on to larger culture flasks.

Passing and maintenance of HeLa and U2OS cells

• The appropriate amount of EDTA-trypsin and complete DMEM in the incubator (37°C), before handling the cells.
• The filter of a 1 ml pipette is broken off, and the pipette is attached to the vacuum suction maschine. The medium is removed, and the pipette is put in a 50 ml vial for later use.
• 1 ml 0,05% EDTA-trypsin/PBS is added to wash the cells. Tilt the flask quickly, so the EDTA-trypsin covers the cells. Remove the liquid quickly.
• 1 ml 0.05% trypsin-EDTA/PBS is added and the flask is incubated for 3 min at 37°C and 5 % CO2.
• 9 ml complete medium is added to inactivate the EDTA-trypsin and to wash the cells of the surface. Make sure the cells are well resuspended, before you transfer them.
• 2,5 ml (depends on the concentration of the cells) cell suspension is transferred to a new 75 cm2 culture flask.
• The cells are kept at 37°C in a 5% CO2 incubator until they are 80-100% confluent. This takes about 2-3 days. Then they are passed on to a new flask or plate.

Transferring the cells to coverslips

• The filter of a 1 ml pipette is broken off, and the pipette is attached to the vacuum suction maschine. The medium is removed, and the pipette is put in a 50 ml vial for later use.
• 1,5 ml 0,05% EDTA-trypsin/PBS is added to wash the cells. Tilt the flask quickly, so the EDTA-trypsin covers the cells. Remove the liquid quickly.
• 1,5 ml 0.05% trypsin-EDTA/PBS is added and the flask is incubated for 3 min at 37°C and 5 % CO2.
• 9 ml complete medium is added to inactivate the EDTA-trypsin and to wash the cells of the surface. Make sure the cells are well resuspended, before you transfer them.
• Calculate the concentration of cells you want in the wells
• Cover slips are placed in the bottom of a 6-well plate (2 cover slips/well). 2 ml cell suspension is added gently to each well.
• The plate is placed in the incubator at 37C and 5% CO2 O/N.