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 :

Plasmidic DNA Extraction on the liquid cultures made Sunday by Nassim and Wilhelm (Zinebe)
--> Followed protocol: “Monarch plasmid miniprep kit” from NEB
Note: First centrifugation of 10 mL, but only 5ml needed, so we re-suspended them in 5mL and centrifuge again.
DNA concentration obtained using Nanodrop
Plasmid Concentration (µg/mL) 260/280 260/230
pMAD 75.136 1.969 2.556
pBS1C 81.240 1.725 0.740

Competency test check (Djamila) done on Friday 19th failed (Boys checked on Saturday) ie bacteria but no rfp from igem plasmid next step?

BSU9716 Plates from Sunday 20th(Wilhelm/Nassim)
No growth on Kan/Cm and Amp, plate back on Kan, Cm and Amp individually (from “loan plate”) and also frozen from initial BSU batch on LB then 37 degrees overnight

Oligo dilution in TE 1X (Sofiane) final concentration 100 uM, at -20

BSU9716 Plates from Monday 20th(Djamila)
Cells on Kan but not on Amp and Cm; should had have it on Kan and Cm so don’t use anymore “loan plate” as BSU9716 source.
Frozen on LB grew, so put it (chose 4 isolates and 2 locations on loan) on Kan/Cm and 37C overnight (Sofiane)

Made more LB agar 15g agar 1L LB

Liquid solutions (not from isolates but loan for bsu and directly from tube in which came from) of pEN and BSU9716 in 10 mL of LB gent (10 ug/mL) and Kan/Cm (50//25ug/mL), respectively, for tomorrow and plated both on appropriate antibiotic

Preparing lysozyme aliquot (Zinebe) :

Concentration = 20g/L :
→ 0,0220 g lysozyme in 1,1 mL miliQ water (mix with vortex)

Conservation dans le congélo -20°C

pEN plasmid extraction (Djamila)
--> Using the “Monarch Plasmid Miniprep Kit”, according to the NEB protocol
Note: Mistakenly eluted with 130 uL instead of 30 uL

Plasmid Concentration (µg/mL) 260/280 260/230
pEN-Gent 33.197 1.872 1.515

Plates from 24/08/16 (Djamila)
Bsu9716 no isolates and pEN single colonies, but on both plate there is a lot of bubbles

gDNA extraction ()

plasmids glycerol stock ()

gBolcks/biobrick resuspension ()

gblocks digestion?

E. coli DH5alpha Transformation with pBS1C using the following protocol :

1. Put 1μL of circular plasmid or all of a ligation reaction of plasmid DNA in a microtube. Gently add ~100μL of competent cells. Do NO DNA control tube with cells and no DNA.
2. Incubate for 30 mins on ice.
3. Heat shock for 1 mins @ 42°C. Put back on ice.
4. Add 900 μL of LB to tubes. Incubate @ 37°C for 30 mins.
5. Plate 490 μL of the cells in LBAmp (100µg/ml plates). Plate 100 μL of the NO DNA control in a LB plate (to check for quality of cells). Grow O/N.

--> 2 boites de LBamp à 100 µg/mL avec du e.coli DH5 alpha + pBS1C
--> 1 boite de LB sans Amp avec que du e.coli DH5 alpha

Transformation from 26/08 worked: Red on transformed and nothing red on negative control

Made 2 bottles of 1X TAE (100 mL TAE 10X and 900 mL water osmosee)

Set up PCR programs for deleted fragments amplification

Week 5 :

Gradient PCR for pEN plasmid (Gent amplification)
61.6 degrees middle and +2 gradient 3 on left and 3 on right tube 4 in whole 7
Dilution Primers: final concentration 5 uM (2.5 uL primer+47.5 uL nuclease free water)
Dilution pEN: 1/10 (2 uL pEN + 18 uL Elution Buffer EB) final concentration 3.32 ug/mL

Component 20 μl Reaction Final Concentration
Nuclease free water 3.9 μl
2X Phusion Master Mix 10 1x
5 μM Forward Primer 2 0.5 μM
5 μM Reverse Primer 2 0.5 μM
Template DNA (3.3 ug/mL or ng/uL) 1.5 5 ng
DMSO (optional) 0.6 3%

Program Gent: IGEM1601

Step Temperature Time
Denaturation 98°C 30s
Annealing and elongation 98°C 30s
61.6°C_ Gradient 2 °C 15s
72°C 15s
Final elongation 72°C 5 min
Hold 4°C -

BSU9716 liquid culture prep

1.2% Gel of Gent amplification PCR product (Djamila)
1.2 g Agarose 100 mL TAE 1X 10 uL (10000 X) Gel red

Samples:
5 uL DNA, 4 uL loading dye, 11 uL MilliQ H2O Run 90V 45 min then 120V 55 min

Gent resistance gene size: 534 bp Band looks like it’s around the expected size
M 1 2 3 4 5 6 7 ADD PICTURE
Note: Have same amount of DNA in all well and this was supposed to be a gradient PCR.
Therefore, it looks like all temperature are working well. Next PCR simple instead of gradient then if don’t work will do gradient
Run PCR for fragment A and B
Component 20 μl Reaction Final Concentration
Nuclease free water 4.4 μl
2X Phusion Master Mix 10 1x
5 μM Forward Primer 2 0.5 μM
5 μM Reverse Primer 2 0.5 μM
Template DNA (initial concentration?) 1 Don’t know how much DNA
DMSO (optional) 0.6 3%

Program A IGEM1602

Step Temperature Time
Denaturation 98°C 30s
Annealing and elongation 98°C 30s
66.2°C 15s
72°C 15s
Final elongation 72°C 5 min
Hold 4°C -

Program B: IGEM1603 65.9 (remember to change the name it’s IGEM1604 on the thermos cycler)

Step Temperature Time
Denaturation 98°C 30s
Annealing and elongation 98°C 30s
65.9 °C 15s
72°C 15s
Final elongation 72°C 5 min
Hold 4°C -

Note: issue with PCR of B; when looked at the TC to check evolution there was an error and we assumed the beginning of the PCR was done so we just did the final elongation step.
Run gel for fragment A and B (second half of the 1.2% Gel of the morning)
3 uL of DNA + 4 uL of loading dye + 13 uL milliQ H2O
Run 140V 30 min 160V 30 min
Band around 500 bp for A and nothing for B. Not surprising since maybe no PCR was done on it. Start over the B tomorrow and join A and Gent. Maybe purify do bigger volume and purify PCR before joining them.

M A B M A B ADD PICTURE
31/08/16 PCR Fragments A, B and Gent
Used diluted samples and oligos done for previous PCR
Component 60 μl Reaction Final Concentration
Nuclease free water 13.2 μl
2X Phusion Master Mix 30 1x
5 μM Forward Primer 6 0.5 μM
5 μM Reverse Primer 6 0.5 μM
Template DNA (initial concentration?) 3 Don’t know how much DNA
DMSO (optional) 1.8 3%
PCR programs: A IGEM1602, B IGEM1603 and Gent IGEM1601 without gradient

1.2% Gel (prepared the 30/08)
5 uL of DNA + 3.5 uL of loading dye + 11.5 uL milliQ H2O
Have a band for each sample around 500 bp as expected but faint for B, and even for A compared to yesterday’s results.

M A B GADD PICTURE
PCR purification
Used 40 uL of each sample. Followed QIAquick PCR Purification Kit Protocol.
Note: Samples centrifuged at 12000 rpm


Recombinant PCR AG
Used diluted samples and oligos done for previous PCR
Component 60 μl Reaction 1 60 μl Reaction 2 Final Concentration
Nuclease free water - -
2X Phusion Master Mix 30 30 1x
5 μM Forward Primer DF1A 6 6 0.5 μM
5 μM Reverse Primer RR 6 6 0.5 μM
Template DNA A 6.6 8.2 (should have been 9.2) ?
Template DNA Gent 6.6 6 ?
DMSO (optional) 1.8 1.8 3%

PCR programs: A-Gent IGEM1601 without gradient (Ta= 61.6)

Recombinant PCR AGB
Used purified AG 1 and 2

Component 60 μl Reaction 1 60 μl Reaction 2 Final Concentration
Nuclease free water 1.2 1.2 -
2X Phusion Master Mix 30 30 1x
5 μM Forward Primer DF1A 6 6 0.5 μM
5 μM Reverse Primer RR 6 6 0.5 μM
Template DNA A 6 6 ?
Template DNA Gent 6 6 ?
DMSO (optional) 1.8 1.8 3%
PCR programs: A-Gent IGEM1604 (Ta= 61.8)

PCR purif (Zinebe)
Used 40 uL of each sample (AG1, 2 and AGB 1, 2). Followed QIAquick PCR Purification Kit Protocol.
Note: Samples centrifuged at 12000 rpm

Gel electrophoresis (Djamila)
5 uL DNA, 4 uL loading dye, 11 uL MilliQ H2O Run 90V 45 min then 120V 55 min

No A-G1 AG2 M AGB1 AGB2
Note: none of the bands are where they are supposed to be. Plan was to ligate into plasmid but first start over gel and check bands. After that may need to start over recombinant PCR

02/09/16 M1 Meeting at UPMC with IONIS

PCR purified samples concentration (Djamila): all are in ng/uL
AG1 113.1
AG2 60
AGB1 20
AGB2 43.5
A 19.8
G 35.6
B 13.4
Note:
• Did the blank with water as there was no EB at Peronnet’s (elution in EB)
• No idea on purity (took pictures to have all purity on file but don’t have the phone anymore and the picures with)

Biobricks digestion ligation Gel

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