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Week 01: 3th - 9th June
Monday 3th June
Tuesday 4th June
Wednesday 5th June
Thursday 6th June
Friday 7th June
Week 02: 10th - 16th June
Monday 10th June
Tuesday 11th June
Wednesday 12th June
Thursday 13th June
Friday 14th June
Week 03: 17th - 23th June
Monday 17th June
Tuesday 18th June
Wednesday 19th June
Thursday 20th June
Friday 21th June
Week 04: 24th - 30th June
Monday 24th June
Tuesday 25th June
Wednesday 26th June
Thursday 27th June
Friday 28th June
Week 05: 1st - 7th July
Monday 01st July
Drug Screening
Media was made, Strains sD001-sD004 were received, inoculated, and put into stock and catalog.
4 strains were received from Jake:
E. coli: BL21 (DE3) ko20 ΔcysI, Δfpr, ΔydbK
E. coli: NEBTurbo zmSIR Chloramphenicol
E. coli: NEBTurbo zmFNR Spectinomycin
E. coli: NEBTurbo soFD, zmSIR Chloramphenicol
Media and Glycerol stock were prepared:
Media Preparation
3 500ml bottles of LB broth and LB agar were prepared by standard methods. 12.5g/500ml powder/water for broth and 20g/500ml powder/water for agar. Bottles were autoclaved. Additionally 1 flask of 500ml of LB broth was made in the same fashion.
Glycerol Stocks
Single colonies from agar plates were picked and used to innoculate 5ml LB broth overnight. 750ml of overnight culture was added to 250ml of 60% glycerol in a cryotube. Two sets of Glycerol stocks were used for each of sD001, sD002, sD003, sD004; one set was frozen at -20ºC and the other set was frozen at -80ºC.
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Tuesday 02st July
Drug Screening
Plasmids were extracted from strains sD002, sD003, sD004
Plasmid Extraction
Plasmids pD004 (pCDF.ew12 zmFNR Spectinomycin), pD005 (pACYC.ew13 soFD, zmSIR Chloramphenicol), pD006 (pACYC.ew17 zmSIR Chloramphenicol) were extracted from NEBTurbo cells using a Thermo Scientific GeneJet Plasmid mini prep kit as described in the protocol (available on google drive). Lacking Resuspention solution we used some from a different mini prep upstairs. Plasmids were eluted in 100ul of nanopure water and frozen at -20ºC. Plasmids were included in catalog.
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Wednesday 03st July
Drug Screening
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Competent Cells : BL21 (DE3) dCysI dFpr dydbk
We grew 5ml of sD001 cells overnight from a single colony.
This 5ml was used to innoculate 500ml of LB broth.
Broth was incubated for 1.5h and optical density was read at 0.62 OD600.
Cells were alloquoted into 10 falcon tubes (50ml each) and centrifuged at 4000xg for 20 minutes at 4C.
Supernatant was removed and cells were resuspended in 200ml of Buffer 1 (4x 50ml) and left on ice for 20 minutes. Cells were centrifuged again for 20 minutes at 4000xg.
Supernatant was removed and cells were resuspended in 12ml Buffer 2.
Cells were alloquoted at 500ul into microcentifuge tubes and frozen at -80C.
Buffer 1: 50mM CaCl2
Buffer 2: 0.53ml 2M CaCl2
2.8ml 60% Glycerol
8.67ml sterile H2O
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Thursday 04st July
Drug Screening
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Friday 04st July
Drug Screening
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Week 06: 8th - 14th July
Monday 8th July
Drug Screening
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Tuesday 9th July
Drug Screening
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Wednesday 10th July
Drug Screening
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Thursday 11th July
Drug Screening
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Friday 12th July
Drug Screening
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Week 07: 15th - 21th July
Monday 15th July
Drug Screening
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Tuesday 16th July
Drug Screening
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Wednesday 17th July
Drug Screening
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Thursday 18th July
Drug Screening
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Friday 19th July
Drug Screening
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Week 08: 22th - 28th July
Monday 22th July
Drug Screening
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Tuesday 23th July
Drug Screening
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Wednesday 24th July
Drug Screening
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Thursday 25th July
Drug Screening
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Friday 26th July
Drug Screening
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Week 09: 29th July - 4th August
Week 10: 5th - 11th August
Week 11: 12th - 18th August
Week 12: 19th - 25th August
Week 13: 26th August - 1st September
Week 14: 2nd - 8th September
Week 15: 9th - 15th September
Week 16: 16th - 22th September
Week 17: 23th - 29th September
Week 18: 30th September - 6th October
Week 19: 7th - 13th October
Week 20: 14th - 20th October
Week 21: 21th - 27th October
Week 22: 28th October - 3th November