Revision as of 16:19, 4 July 2016

Wednesday 29^th 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 abs_600nm=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

K1372001 plasmid was digested with BglI using the following quantities: {

@@ Line 1: / Line 1: @@
 {{Team:Paris_Saclay/notebook_header}}
-=Wednesday 29th June=
+=Wednesday 29<sup>th</sup> June=
 ==Lab work==
@@ Line 7: / Line 7: @@
 ''By Lea and Marion''
-We got 6 clones of each construction in 3,5ml LB (50 µg/ml Amp). Incubation overnight at 37°C, 180rpm.
+clones of each construction were selected and grown overnight in 3.5mL of LB (50µg/ml Amp) at 37°C, 180rpm.
@@ Line 14: / Line 14: @@
 ''By Charlene''
-We added 200µL of BL21 pre-culture in 15mL of LB. It incubated 4h at 37°C. When abs600nm=0,68 cells have been centrifuged 10 minutes at 4000rpm. We did 2 washes with addition of 10mL glycerol (10%) followed by 10 minutes of centrifugation at 4000rpm. We putted cells in 200 µL glycerol.
+µL of BL21 pre-culture was added to 15mL of LB and incubated for 4h at 37°C. When abs<sub>600nm</sub>=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
+µL 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. {{Font color||yellow|Value = 1.6 What is this value???}}. Cells died during transformation.
+Transformation worked for K1372001+pcl_TAG and K1372001+pcl_Tq. {{Font color||yellow|Value = 6}}.  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====
+K1372001 plasmid was digested with BglI using the following quantities:
+{
 {{Team:Paris_Saclay/notebook_footer}}

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