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Team:Evry/Notebook/w3
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<h1>Week 3: 25th June - 1st July</h1> | <h1>Week 3: 25th June - 1st July</h1> | ||
Latest revision as of 09:57, 3 August 2012
Weeks:
| June | July | August | September | October | November | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Week 3: 25th June - 1st July
Monday, 25th June
Bacterial Transformation
Bacteria:T10Reporters:
- RFP: P1-18F K
- GFP: P1-14K A
- CFP: P1-6A A
- YFP: P2/24E AK
- OFP: P2/13N K
- Violecein P3/12B T
- Vio operon ABDE P3/20H K
- Vio operon ABCE P3/20J K
- CelEBI w/RRS P3/6H A
- CelEBY w/RRS P3/6L H
- Keep constantly the cells on ice
- Rehydratation of DNA in 10uL H2O 2) Add 1 uL of DNA in 50 uL of T10
- Incubate 30 min on ice
- Heat shock the cells during 30 sec at 42 degree Celsius in a water-bath without shaking
- Put 2 min on ice
- Add 500 uL of pre warmed SOC-medium
- Incubate 1h at 37 degree Celsius at 225 rpm
- Spin at 5000 rpm during 30 sec
- Remove 150 uL - 400 uL of supernatant
- Resuspend the pellet in the 150 uL left
- Spread on appropriate plates
- Incubate overnight at 37 degree Celsius
Tuesday, 26th June
Bacterial Transformation:
Petri dish- YFP
- GFP
- CFP
- RFP
- pCS2 (+) from 22/06/12
Wednesday, 27th June
- MiniPrep- followed by nanodrop: confirmation of DNA presence in transformed bacteria
- Agarose gel electrophoresis (samples of 4 fluorescent DNA)
- Inoculation of colonies in LB medium- incubation at 37 degrees overnight
Thursday, 28th June
Plasmid purification:
Protocol:- Pellet 3mL bacterial culture by centrifugation at 8000 rpm for 3 min at room temperature
- Resuspend pelleted bacterial cells in 250 uL Buffer P1
- Add 250 uL Buffer P2 and mix 4-6 times. Let it for no more than 5 min
- Add 350 uL Buffer N3 and mix 4-6 times
- Centrifugation at 13 000 rpm for 10 min
- Take the supernatant and apply to the QIAprep spin column
- Centrifugation at 13 000 rpm for 60 sec
- Discard flow-through
- Centrifugation at 13 000 rpm for 60 sec
- Discard flow-through
- Place column in a clean 1.5 mL microcentrifuge tube
- Add 50 uL Buffer EB to the column for elution and let stand for 1 min
- Centrifugation at 13 000 rpm for 1 min
DNA Concentrations:
| Type | Concentration (ng.uL-1) |
|---|---|
| pCS2 | |
| CFP | |
| GFP | |
| RFP | |
| YFP | |
MiniPrep digestion:
CFP, GFP, RFP, YFP : EcoRI/SpeI(BcuI)- Supermix: 10uL buffer Red + 1uL BSA + 74uL
- Mix for each sample : 21,25uL supermix + 1uL DNA + 0,5uL EcoRI + 0,5uL BcuI
- 2,5uL buffer Orange + 0,25uL BSA + 18,5uL ddH2O
- 3h, 37°C
Friday, 29th June
Gel migration 1
- Gel preparation: 25mL agarose+TAE (freezer) + 2microL BET
- Sample preparation:
- DNA Ladder 1kB: 1microL ladder + 1microL loading buffer + 4microL ddH2O
- Digested RFP, YFP, CFP, GFP, pCS2+ (from 28 june): 10microL sample + 2microL loading buffer
- 100V, 1h
DNA digestion:
- 4ul of CFP, GFP, RFP, YFP dosed on 28 june
- 3ul of pSC2+ dosed on 28 june
Gel migration 2:
- Gel preparation: 50mL agarose+TAE (freezer) + 4ul BET
- Sample preparation:
- DNA Ladder 1kB: 1ul ladder + 1ul loading buffer + 4ul ddH2O
- Digested RFP, YFP, CFP, GFP, pCS2+ (from 29 june) : 15ul sample + 3ul loading buffer
- 100V, 1h
