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From 2012.igem.org

Weeks

1

2

3

4

5

6

7

8

9

10

11

12

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14

15

16

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

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 (see picture below):

Friday, 29th June

Retrieved from "http://2012.igem.org/Team:Evry/Notebook/w3"