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INSA-Lyon/23 September 2010
From 2010.igem.org
September 23rd
DNA extraction of I0500, 4O, 10L, 8C/14K (2 clones) ; Agarose gel electrophoresis for verification :
There is no more plasmide for 4O, 10L and I0500. We will ask some new.
Transformation of the ligation overnight of yesterday
Measure of fluorescence and DO of the liquid culture of PHL1273 of the 20/09. Start of 3 new cultures in a waterbath at 37°C with a shaking speed of 100 rpm
Competent cells production of PHL818 strain: 500µl overnight culture in 100ml LB-Tet, at 37°C until DO600nm=0,49. Chemiocompetence had been done with protocol described on wiki. 12 competent bacteria vials was stored at -80°C.
4 plasmid DNA extractions (14K (Amp), I0500 (Kan), 1F/24A (Tet) and 18A/24A (Cm)) with NucleoSpin Plasmid kit (Macherey-Nagel) with four different antibiotic resistances. Goal of the experiment: production of plasmid backbones.
Lyophilisated primers SB-prep-3P and 2Ea are diluted in sterile water (at 100µM): 3P with 325µl and 2Ea with 356µl.
5 PCR had been done in order to produce iGEM backbones: vial 1 corresponded to the negative control with 10µl sterile water, vial 2 corresponded to the plasmid Amp, vial 3 corresponded to the plasmid Kan, vial 4 corresponded to the plasmid Tet and vial 5 corresponded to the plasmid Cm.
MIX composition for one PCR:
- 5µl 10X buffer with MgCl2
- QSP H2O
- 5µl dNTP (PCR Dig Labeling Mix, Roche)
- 10µl plasmid DNA (diluted 1/5)
- 1µl of each primer
- 0,75µl Taq Pol (Euromedex)
NB: Hybridation temperature : 58°C (program PSB1)
Gel verification (0,7%) without ladder (no more!!): Negative control OK, Amp: too many strips (no strip at the expected size), Kan: no DNA (I0500 plasmid extraction never gives DNA, it’s confirmed), Tet et Cm: the two biggest strips seem at the good sizes (2,5 and 2kb). PCR products and diluted primers are stored at -20°C.
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