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Team:Cambridge/LabBook/Week7
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| + | ==Monday== | ||
===51. Experiment: Transformation with pJS555 & pSB1C3 (Theo and Hannah)=== | ===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)). | 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)). | ||
| Line 4: | Line 5: | ||
Theo transformed TOP10 with pSB1C3 from registry. | Theo transformed TOP10 with pSB1C3 from registry. | ||
| - | ===52. Experiment: | + | ===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 | ||
Revision as of 14:23, 27 August 2010
Contents |
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
