Difference between revisions of "Team:KU Leuven/Research/Methods"

Make a liquid culture of a single colony in 1-3 mL salt free LB
Grow 300-400 mL cells (without salt) in 37°C until the O.D.reaches 0.6
Cool down on ice and from now on 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). You should have a pulse of 4-6 msec. If it is shorter, wash the cells once again with 30 mL glycerol
Aliquot the cells (50 µL) and 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 this out on pre-warmed plates (37 °C)
J23101, J23106 and J23117 were plated out on chloramphenicol and I13504 was plated out on ampicillin

Digest I13504 (GFP) with XbaI and PstI in the 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 visualise 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 a Tango buffer
Mix gently and spin down
Put this for 2 hours at 37 °C in a heating block
Gel electrophoresis of the samples in 1.5% agarose gel
Analyse the gel picture and interpret the results

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