Newcastle University Wetlab/12 September 2008
From 2008.igem.org
Newcastle University
GOLD MEDAL WINNER 2008
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Wet lab work was carried out from 4 August to 19 September, Mondays to Fridays. Please click on a day to see the lab notebook.
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Friday 12th September
- The ON cultures of iGEMgfp and Bs168 were diluted by adding 250μl ON culture to 10mL LB and incubating at 37°C whilst shaking for 2 hours. The two diluted cultures were then each split into 5 x 2mL aliquots and subtilin added in the following concentrations:
- 0% induction (2mL dilute culture)
- 0.1% induction (2mL dilute culture + 2μl subtilin)
- 0.5% induction (2Ml dilute culture + 10μl subtilin)
- 1% induction (2mL dilute culture + 20μl subtilin)
-10% induction (2mL dilute culture + 200μl subtilin)
The five Bs168 tubes acted as controls. These were incubated at 37°C whilst shaking for 1 hour to allow the subtilin to induce gfp expression.
- Plasmid was isolated from the 12 pGFPrrnB-BB107 ON cultures 9see protocol). Unfortunately plasmid from colony 4 (25μl plate) and colony 4 (250μl plate) were lost due to human error.
- pGFPrrnB-ncl08 colony 7 and pJWV021 colony 13 isolated from overnight cultures (see protocol).
- All isolated plasmid was restricted using 7.5μl plasmid and 0.5μl each enzyme in 15μl total volume:
- pGFPrrnB-BB107 restrited with EcoRI and SpeI.
- pGFPrrnB-ncl08 restricted with EcoRI and NheI
- pJWV021-ncl08 restricted with NheI and BglII
These restrictions were run on gels.
Lane 1: 1kb ladder
Lane 2: pGFPrrnB-BB107 colony 1
Lane 3: pGFPrrnB-BB107 colony 2
Lane 4: pGFPrrnB-BB107 colony 3
Lane 5: pGFPrrnB-BB107 colony 5
Lane 6: pGFPrrnB-BB107 colony 6
Lane 7: pGFPrrnB-BB107 colony 7
Lane 8: pGFPrrnB-BB107 colony 8
Lane 9: pGFPrrnB-BB107 colony 9
Lane 10: pGFPrrnB-BB107 colony 10
Lane 11: pGFPrrnB-BB107 colony 11
Lane 1: 1kb ladder
Lane 2: pGFPrrnB-ncl08 colony 7
Lane 3: pJWV021-ncl08 colony 13
Lane 4: pGFPrrnB-ncl08 colony 7 control (no enzyme)
Lane 5: pJWV021-ncl08 colony 13 control (no enzyme)