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From 2010.igem.org

Contents 1 Monday 1.1 51. Experiment: Transformation with pJS555 & pSB1C3 (Theo and Hannah) 1.2 52. Experiment: Check for promoter+rbs+p.p.luc - obtain from the colony from transformation on page 37. (Paul) 1.2.1 Colony PCR 1.2.2 Gel Electrophoresis

Monday

51. Experiment: Transformation with pJS555 & pSB1C3 (Theo and Hannah)

Received a new plasmid from Jim Slock, so have transformed some chemically competent cells with it to build up a stock and test it later/we think it will glow for longer than the previous one (says Mr. Lovely)).

Theo transformed TOP10 with pSB1C3 from registry.

52. Experiment: Check for promoter+rbs+p.p.luc - obtain from the colony from transformation on page 37. (Paul)

Colony PCR

Take sample from colony with loop and put in 20μl of water to obtain DNA template.

Then prepare on isoFreeze PCR chiller in lister order:

• 10μl of 2xPhusion Master Mix
• 1μl of VR
• 1μl of VF2
• 1μl of DNA template
• 7μl of Nuclease-Free H20

Then PCR:

1. 98°C for 15 mins

2. 98°C for 10 secs

3. 65°C for 30 secs

4. 72°C for 1 min

5. repeat 2-4 35 times

6. 72°C for 10 mins

7. 4°C forever

Results:

Tube 1: 197.4ng/μl

Tube 2: 235.3ng/μl

Gel Electrophoresis

• Tube 1: 194.7ng/μl
  • 4μl of DNA
  • 3μl of 6x Orange Loading Dye
  • 13μl Nuclease Free H20

• Tube 2: 235.3ng/μl
  • 3μl of DNA
  • 3μl of 6x Orange Loading Dye
  • 14μl of Nuclease Free H20