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Revision as of 12:15, 18 October 2016
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 :
- ➟10mL 1M Tris pH 7.5 + 2mL 0,5M EDTA pH 8.0
- ➟0,5M EDTA : 18,6g EDTA + 100 mL H2O. Ajuste pH 8.0 with solid NaOH (2,6g)
- ➟1M Tris : 30,285g + 205 mL H2O. Ajust pH 7.5 with HCl.
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 :
- ➟50% (w/v) glucose 2 ml
- ➟1.2% (w/v) MgSO4·3H2O 14 ml
- ➟10% (w/v) Bacto yeast extract 2 ml
- ➟1% (w/v) casamino acids 2 ml
- ➟4 ml EGTA (0.1 M, pH 8.0)
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
- Benjamin : Il s'est fait chier
- Nassim : Il a cassé les couilles
- Sofiane : Il a tout fait cramer
- Wilhelm : Il est reparti
- Kévin : Il est pas venu
- Perwin : Continuation de la quête chez les RG
- Zinebe : Elle a volé le PLA
- Djamila : Comme d'hab, le quota
- Les autres : Vacances
Week 5 :
Ca me soule toujours autant
- Benjamin : Il s'est fait chier
- Nassim : Il a cassé les couilles
- Sofiane : Il a tout fait cramer
- Wilhelm : Il est reparti
- Kévin : Il est pas venu
- Perwin : Continuation de la quête chez les RG
- Zinebe : Elle a volé le PLA
- Djamila : Comme d'hab, le quota
- Les autres : Vacances
Week 6 :
Ca me soule toujours autant
- Benjamin : Il s'est fait chier
- Nassim : Il a cassé les couilles
- Sofiane : Il a tout fait cramer
- Wilhelm : Il est reparti
- Kévin : Il est pas venu
- Perwin : Continuation de la quête chez les RG
- Zinebe : Elle a volé le PLA
- Djamila : Comme d'hab, le quota
- Les autres : Vacances
Week 7 :
Ca me soule toujours autant
- Benjamin : Il s'est fait chier
- Nassim : Il a cassé les couilles
- Sofiane : Il a tout fait cramer
- Wilhelm : Il est reparti
- Kévin : Il est pas venu
- Perwin : Continuation de la quête chez les RG
- Zinebe : Elle a volé le PLA
- Djamila : Comme d'hab, le quota
- Les autres : Vacances
Week 8 :
Ca me soule toujours autant
- Benjamin : Il s'est fait chier
- Nassim : Il a cassé les couilles
- Sofiane : Il a tout fait cramer
- Wilhelm : Il est reparti
- Kévin : Il est pas venu
- Perwin : Continuation de la quête chez les RG
- Zinebe : Elle a volé le PLA
- Djamila : Comme d'hab, le quota
- Les autres : Vacances