Difference between revisions of "Team:KU Leuven/InterLabStudy/Protocol"

Make a liquid culture of a single colony in 1-3 mL salt free LB
Grow 300-400 mL cells (without salt) at 37°C until the O.D. reaches 0.6
Cool down on ice and perform all the steps at 4°C
Spin the cells down in falcon tubes (3500 g, 20 min, 4°C)
Resuspend the cells in 10% glycerol, spin the cells down (5000 g, 10 min, 4 °C). Repeat this step 3 times
Resuspend the cells in 10% glycerol to obtain a dense pulp (usually not more than 1.5 mL)
Take 50 µL sample and do the electroporation test (without DNA). Pulses should be between 4 and 6 msec. If shorter, wash the cells once again with 30 mL glycerol
Aliquot the cells (50 µL), quick-freeze in liquid nitrogen and store at -80°C

Add 1 µl DNA to 50 µl electrocompetent cells in an ice-cold cuvette (1 mm)
Electroporate (Eppendorf, 1700 V, 4 msec)
Add 950 µl of SOC solution
Incubate for one hour at 37°C
Plate out on pre-warmed plates containing the correct selective medium, in this case chlormaphenicol for J23101, J23106 and J23117 and ampicillin for I13504 (37°C)

Digest I13504 (GFP) with XbaI and PstI in Tango buffer
Digest the promoters J23101, J23106 and J23117 with PstI in buffer O
Load the digested I13504 on a 1.5% agarose gel and visualize it under UV light. Thereafter, perform a gel purification of I13504 (GeneJET Gel Extraction Kit - ThermoFisher Scientific)
PCR purify the promoters J23101, J23106 and J23117
Digest the promoters J23101, J23106 and J23117 with FD SpeI in 10x Fast Digest Buffer
Ligate every promoter with I13504 using T4 DNA ligase

Digest with NcoI (cuts 1x in pSB1C3) and XhoI (cuts 1x in GFP) in Tango buffer
Mix gently and spin down
Incubate for 2 hours at 37°C in a heating block
Separate the fragments using gel electrophoresis on a 1.5% agarose gel