Difference between revisions of "Team:UPMC-Paris/Notebook"

Week 1 :

Competence of Bsu9716:

Bacteria from 2/8/16 in fridge (Dj)
50 ml LB and 10 uL Kan added (so total 150 mL and 15 uL Kan) and cells back in incubator
Spin (4000 rpm 10 min) 50 ml X 2 and used pellet for competency
Spinned again same way after pulling both pellet together
Changed medium (50 mL LB + 5 uL Kan) and put it back at 4 degrees

Stock solutions (Dj)

Name Mass (g) Total Volume (mL)
1.2% MgSO4 0.1023 12.5
10% (w/v) Bacto yeast extract 1.0096 10
1% (w/v) casamino acids 0.1039 10
0.1 M CaCl2 (110.98 g/mol) 0.1792 15

SpC medium made by Djamila
T base 20 ml
50% (w/v) filtered glucose 0.2 ml
1.2% (w/v) MgSO4 0.3 ml
10% (w/v) Bacto yeast extract 0.4 ml
1% (w/v) casamino acids 0.5 ml

ODi 0.5 and put back at 37 degrees with shaking
ODf 0.8

SpII medium made by Sofiane
T base 200 ml
50% (w/v) filtered glucose 2 ml
1.2% (w/v) MgSO4 14 ml
10% (w/v) Bacto yeast extract 2 ml
1% (w/v) casamino acids 2 ml
0.1 M CaCl2 1 ml

2 mL of SpC BSU in SpII and 90 min incubation 37 degrees with shaking
Note:
Originally 1.2% of the dihydrate form so made the calculations in function of that; and Sofiane had to make a second batch
50% (w/v) glucose was filtered
No tryptophan added for the cell growth

Plates from 03/08/2016 (Zinebe)
Contains lawn and colonies ⇒ Not sure which are the B. subtilis.
Diluted some of the lawn and the colony each in 200 uL LB and plated it on LB-Kan then 37 degrees overnight

Note:
LB used for dilution was contaminated
When picking the colony with a tip some of the lawn was scraped with it

Pick primers and autoclaved MilliQ at school

Plates (from 04/08/16) (DJ)
Both plates had lawns on them however the plate containing the colony (along with some lawn and labelled Isolate) was green which was weird

Took plate from 3/8/16 and streaked on a plate divided in 3 a single colony, the lawn, and for the last portion, 10 uL of the B subtilis solution in the fridge was diluted in 200 uL of LB before streaking it.
The rest of B subtilis solution in the fridge was spinned, diluted in 10ml LB and divided in 2 x 50 mL tubes, 5 uL of Kan was added in each. Both were put back in the fridge

Tris HCl made by Kevin

All streaks grew well so we put them in fridge

Week 2 :

Nothing done at the bench this week because of material issues

Week 3 :

Tris-EDTA preparation (Sofiane):

1. ➟10mL 1M Tris pH 7.5 + 2mL 0,5M EDTA pH 8.0


2. ➟0,5M EDTA : 18,6g EDTA + 100 mL H2O. Ajuste pH 8.0 with solid NaOH (2,6g)


3. ➟1M Tris : 30,285g + 205 mL H2O. Ajust pH 7.5 with HCl.

Note : pH ajustment failed, I add 25mL of 12% HCl and 12 mL of 20% HCl to have a pH 7,6.
Concentration is not good anymore. We have to make this solution again. Maybe buy TE Buffer? Verification of TE we have is needed!

Competency test (Djamila/Zinebe)
Chloramphenicol (cm 25 mg/mL) preparation:

0.25 g diluted in 10 mL EtOH 95%; aliquots at -20

Note: first dilution in milliQ water by mistake; got rid of water and tried to let it dry but without success so will probably just add directly EtOH in to make more Cm

Cm (Cf = 5 ug/mL) LB plates:
70 mL melted LB agar + 14 uL cm
Note: agar cooled too much before pouring plate (only got 3 plates) so made more

100 mL melted LB agar + 20 uL cm

Competency test (CT)

Made SpII-EGTA solution (solutions made yesterday by Sofiane)

T base 200 ml :

1. ➟50% (w/v) glucose 2 ml
   
2. ➟1.2% (w/v) MgSO4·3H2O 14 ml
   
3. ➟10% (w/v) Bacto yeast extract 2 ml
   
4. ➟1% (w/v) casamino acids 2 ml
   
5. ➟4 ml EGTA (0.1 M, pH 8.0)
   

Aliquots put at -20°C

After thawing competent BSU9716 at 37C in incubator (water bath had issues), added 500 uL of SpII-EGTA and mixed up and down.
Added 50 uL bacteria in 5 eppendorf tubes after spinning CT plasmids, added 1 uL of DNA in the appropriate tube (50pg/ul, 20pg/ul, 10pg/ul, 5pg/ul, 0.5pg/ul).
Bacteria+DNA mixture incubated at 37°C with shaking (150 rpm) for an hour
Spreaded the bacteria on Cm LB agar plate then put at 37°C overnight
At this point from the rest of competent bacteria, 100 uL spread on Kan plates

Note:
Should have spread the transformed bacteria on Cm and Kan plates
Competency test kit contains five vials of purified DNA from BBa_J04450 (RFP construct) in plasmid backbone pSB1C3 (cm resistant). Each vial contains DNA at a different concentration: 50pg/ul, 20pg/ul, 10pg/ul, 5pg/ul, 0.5pg/ul.

Week 4 :

Ca me soule toujours autant

1. Benjamin : Il s'est fait chier
2. Nassim : Il a cassé les couilles
3. Sofiane : Il a tout fait cramer
4. Wilhelm : Il est reparti
5. Kévin : Il est pas venu
6. Perwin : Continuation de la quête chez les RG
7. Zinebe : Elle a volé le PLA
8. Djamila : Comme d'hab, le quota
9. Les autres : Vacances

Week 5 :

Ca me soule toujours autant

1. Benjamin : Il s'est fait chier
2. Nassim : Il a cassé les couilles
3. Sofiane : Il a tout fait cramer
4. Wilhelm : Il est reparti
5. Kévin : Il est pas venu
6. Perwin : Continuation de la quête chez les RG
7. Zinebe : Elle a volé le PLA
8. Djamila : Comme d'hab, le quota
9. Les autres : Vacances

Week 6 :

Ca me soule toujours autant

1. Benjamin : Il s'est fait chier
2. Nassim : Il a cassé les couilles
3. Sofiane : Il a tout fait cramer
4. Wilhelm : Il est reparti
5. Kévin : Il est pas venu
6. Perwin : Continuation de la quête chez les RG
7. Zinebe : Elle a volé le PLA
8. Djamila : Comme d'hab, le quota
9. Les autres : Vacances

Week 7 :

Ca me soule toujours autant

1. Benjamin : Il s'est fait chier
2. Nassim : Il a cassé les couilles
3. Sofiane : Il a tout fait cramer
4. Wilhelm : Il est reparti
5. Kévin : Il est pas venu
6. Perwin : Continuation de la quête chez les RG
7. Zinebe : Elle a volé le PLA
8. Djamila : Comme d'hab, le quota
9. Les autres : Vacances

Week 8 :

Ca me soule toujours autant

1. Benjamin : Il s'est fait chier
2. Nassim : Il a cassé les couilles
3. Sofiane : Il a tout fait cramer
4. Wilhelm : Il est reparti
5. Kévin : Il est pas venu
6. Perwin : Continuation de la quête chez les RG
7. Zinebe : Elle a volé le PLA
8. Djamila : Comme d'hab, le quota
9. Les autres : Vacances