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Team:Cambridge/LabBook/Week7
From 2010.igem.org
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
53. Experiment: Extracting glycerol stocks of TOP10 with promoter+rbs+p.p.luc from registry. (Emily and Peter)
1. Take tube of glycerol stock from -80°C 2. Use loop/tooth pick (we used a loop) to streak on ampicillin resistance plate. Let it melt first, then streak.
NB NEVER let glycerol stocks thaw. They should be out and back in the -80°C freezer in 2-3mins max
3. Place in 30°C incubator overnight.
To be continued tomorrow.
54. Check for promoter+rbs+luc from p32 by luciferin injection. (Paul and Ben)
We used protocol described on p22.
3x Freezymes at -80°C, thawing at 37°C
