Team:Chiba/Calendar-Home/15 October 2008
From 2008.igem.org
14 October 2008 <|> 16 October 2008
Laboratory work
Team:Demo-Is
- Pre-culture
- Picked and cultured the following glycerol stocks in 2mL of LB:
- LB-Amp, BBa_T9002, (JW1908)
- LB-Amp+0.2%Glu, BBa_K084012(plac+rbs+LuxI(no LVA)), (XL10G)
- LB-Amp+0.2%Glu, BBa_K084007(plac+rbs+LasI(no LVA)), (XL10G)
- Cultured at 37°C for 12h.
- Picked and cultured the following glycerol stocks in 2mL of LB:
- Culture
- Added 6.25% each of the pre-cultures to new LB medium.
- LB-Amp, BBa_T9002
- LB-Amp+0.2%Glu, BBa_K084012(plac+rbs+LuxI(no LVA)), BBa_K084007(plac+rbs+LasI(no LVA))
- Cultured at 37°C for 4~5h。
- Added 6.25% each of the pre-cultures to new LB medium.
- Wash
- Transfer 10mL each of the culture to 50mL centrifuge tubes.
- Centrifuged for 6min at 3600rpm,20°C and discarded the supernatant.
- Added LB-Amp to each centrifuge tube:
- 10mL to the tube that contains BBa_T9002
- 5mL to the tube that contains BBa_K084012, BBa_K084007
- Centrifuged for 6min, 3600rpm at 20°C the tube containing BBa_K084012, BBa_K084007 and discarded the supernatant.
- 10mL to the tube that contains BBa_K084012, BBa_K084007
- Centrifuged for 6min, 3600rpm at 20°C the tube containing BBa_K084012, BBa_K084007 and discarded the supernatant.
- 5mL to the tube that contains BBa_K084012, BBa_K084007
- Mix
- Mixed the sender cells BBa_K084012 and BBa_K084007 both with BBa_T9002 at a 1:1 ratio.
- Added 100μL each to a 96-well shallow plate (as shown in the figure).
- Green part is BBa_K084012:BBa_T9002=1:1
- Red part is BBa_K084007:BBa_T9002=1:1
- Uncolored part is BBa_T9002 alone.
- Culture and observe results