Newcastle University Wetlab/11 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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Thurday 11th September
- Single colonies of iGEMcherry were streaked from the overnight kanamycin plates onto sucrose and glucose plates.
- Stab cultures were taken from the overnight 25μl and 250μl pGFP-BBa_I7045898 plates (grown on spectinomycin) and grown in 10mL LB:
- 25μl plate white colony 1
- 25μl plate white colony 2
- 25μl plate white colony 3
- 25μl plate white colony 4
- 25μl plate white colony 5
- 25μl plate white colony 6
- 250μl plate white colony 1
- 250μl plate white colony 2
- 250μl plate white colony 3
- 250μl plate white colony 4
- 250μl plate white colony 5
- 250μl plate green colony 6
These were incubated overnight at 37˚C whilst shaking.
- iGEMgfp was diluted by adding 250μl of the overnight culture to 10mL LB in a sterile shake flask. This was incubated at 37˚C whilst shaking for a further 2 hours to give the cells fresh nutrients and allow them to multiply. This culture was then divided into 3 tubes (3mL in each) and subtilin added in the following concentrations:
- 0% induction (3mL dilute iGEMgfp)
- 1% induction (3mL dilute iGEMgfp + 30μl subtilin)
- 10% induction (30mL dilute iGEMgfp + 300μl subtilin)
These were incubated whilst shaking for 1 hour at 37°C. Flow cytometry was then performed on the samples.
- Overnight cultures were made from glycerol stocks pGFPrrnB-ncl08 colony 7 and pJWV021-ncl08 colony 13 (see protocol). The plasmid from these cultures are to be checked for the 2.2kb fragment before being sent for sequencing.