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Line 36: ====K1372001 plasmid digestion====
====K1372001 plasmid digestion====
− K1372001 plasmid was digested with BglI using the following quantities:
+ ''By Naiane''
− {
+
+ K1372001 plasmid was digested with BglI using the following quantities: 3 µL DNA, 2µL buffer (O), 1 µL enzyme (BglI), 14µL water. The mix was incubated for 1h at 37°C and 10 min at 55°C and kept at -20°C until the gel electrophoresis was done. Fragments are expected to size 1.2kb and 2.4kb.
+
+
+ ===Bringing DNA closer===
+ ====Plasmids digestion====
+ ''By Naiane''
+
{{Team:Paris_Saclay/notebook_footer}}
{{Team:Paris_Saclay/notebook_footer}}
Revision as of 16:40, 4 July 2016
Wednesday 29th June Lab work Visualization Culture of clones with gBlocks By Lea and Marion
6 clones of each construction were selected and grown overnight in 3.5mL of LB (50µg/ml Amp) at 37°C, 180rpm.
BioBrick characterization BL21 electro-competent cells preparation By Charlene
200µL of BL21 pre-culture was added to 15mL of LB and incubated for 4h at 37°C. When abs600nm =0.68, cells were centrifuged for 10 minutes at 4000rpm and washed twice with addition of 10mL glycerol (10%) followed by 10 minutes of centrifugation at 4000rpm. Cells were mixed with 200µL glycerol and kept -80°C.
BL21 electro-competent cells transformation By Charlene
We realized four transformations with the following plasmids:
K1372001
K1372001+pcl_TAA
K1372001+pcl_TAG
K1372001+pcl_Tq 50µL of cells were added in the cuvettes with 1µL DNA (diluted 1/10). Everything was done at 4°C.
Transformation did not work for K1372001 and K1372001+pcl_TAA because the cuvettes were broken. Template:Font color . Cells died during transformation.
Transformation worked for K1372001+pcl_TAG and K1372001+pcl_Tq. Template:Font color . We added 1mL of LB to the cells and incubated them for 1h at 37°C.
The following cells were plated on Petri dishes (30µg/mL Cm + 50µg/mL streptomycin):
BL21|K1372001+pcl_TAG : 50µL of transformed cells
BL21|K1372001+pcl_TAG : 500µL of transformed cells concentrated 5x.
BL21|K1372001+pcl_Tq : 50µL cells
BL21|K1372001+pcl Tq : 500µL of transformed cells concentrated 5x. K1372001 plasmid digestion By Naiane
K1372001 plasmid was digested with BglI using the following quantities: 3 µL DNA, 2µL buffer (O), 1 µL enzyme (BglI), 14µL water. The mix was incubated for 1h at 37°C and 10 min at 55°C and kept at -20°C until the gel electrophoresis was done. Fragments are expected to size 1.2kb and 2.4kb.
Bringing DNA closer Plasmids digestion By Naiane
