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From 2012.igem.org

Weeks:

June			July				August				September				October				November

Week 3: 25th June - 1st July

Monday, 25th June

Bacterial Transformation

Bacteria:T10
Reporters:

• 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

Protocol:

1. Keep constantly the cells on ice
2. Rehydratation of DNA in 10uL H2O 2) Add 1 uL of DNA in 50 uL of T10
3. Incubate 30 min on ice
4. Heat shock the cells during 30 sec at 42 degree Celsius in a water-bath without shaking
5. Put 2 min on ice
6. Add 500 uL of pre warmed SOC-medium
7. Incubate 1h at 37 degree Celsius at 225 rpm
8. Spin at 5000 rpm during 30 sec
9. Remove 150 uL - 400 uL of supernatant
10. Resuspend the pellet in the 150 uL left
11. Spread on appropriate plates
12. 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

1. MiniPrep- followed by nanodrop: confirmation of DNA presence in transformed bacteria
2. Agarose gel electrophoresis (samples of 4 fluorescent DNA)
3. Inoculation of colonies in LB medium- incubation at 37 degrees overnight

Thursday, 28th June

Plasmid purification:

Protocol:

1. Pellet 3mL bacterial culture by centrifugation at 8000 rpm for 3 min at room temperature
2. Resuspend pelleted bacterial cells in 250 uL Buffer P1
3. Add 250 uL Buffer P2 and mix 4-6 times. Let it for no more than 5 min
4. Add 350 uL Buffer N3 and mix 4-6 times
5. Centrifugation at 13 000 rpm for 10 min
6. Take the supernatant and apply to the QIAprep spin column
7. Centrifugation at 13 000 rpm for 60 sec
8. Discard flow-through
9. Centrifugation at 13 000 rpm for 60 sec
10. Discard flow-through
11. Place column in a clean 1.5 mL microcentrifuge tube
12. Add 50 uL Buffer EB to the column for elution and let stand for 1 min
13. Centrifugation at 13 000 rpm for 1 min

DNA Concentrations:

Type	Concentration (ng.uL-1)
pCS2	362,7
CFP	240,1
GFP	209,1
RFP	142,8
YFP	196,0

MiniPrep digestion:

CFP, GFP, RFP, YFP : EcoRI/SpeI(BcuI)

1. Supermix: 10uL buffer Red + 1uL BSA + 74uL
2. Mix for each sample : 21,25uL supermix + 1uL DNA + 0,5uL EcoRI + 0,5uL BcuI

pCS2+ : EcoRI/XbaI

1. 2,5uL buffer Orange + 0,25uL BSA + 18,5uL ddH2O
2. 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

Result: no DNA detectable, Solution: to redo the step of digestion with more DNA

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

Result: no RFP detectable (->):

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