IGEM:IMPERIAL/2006/LabCalendar/2006-8-22: Difference between revisions
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==S01656 Testing - AiiA activity test== | ==S01656 Testing - AiiA activity test== | ||
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**Start ligation at 10:20 | **Start ligation at 10:20 | ||
**Gene clean ligation at 12:30, ready for electroporation | **Gene clean ligation at 12:30, ready for electroporation | ||
==Aiia Western Blot== | |||
*Cultured a fresh day culture of S01656 (aiiA test) for a western blot at '''9:30''' | |||
*At '''11.10''' | |||
**Took a 1ml control | |||
**Added 4ul of 1molar IPTG to the remaining 4ml | |||
[[http://openwetware.org/wiki/IGEM:IMPERIAL/Protocols/western_blot western blot protocol]] | |||
[[User:JohnChattaway|JohnChattaway]] 06:17, 22 August 2006 (EDT) | |||
==ImmunoTag PCR== | |||
*Successful PCR!!! All three repeats | |||
*DNA run on gels and cut out and purified using the glass milk technique | |||
*Ligation into T tail overhanging plasmid currently ongoing. Started at 2.50pm. | |||
*4.50pm- Ready for electroporation |
Revision as of 07:41, 22 August 2006
S01656 Testing - AiiA activity test
- OD of overnight cultures
- -IPTG - 0.430
- +IPTG - 0.550
- Recultured into 5 mL as per protocol, shake start at 9:35
- OD of second culture
- -IPTG - 0.355
- +IPTG - 0.285
- Recultured into 5 mL LB medium (no kanamycin), separated cultures into eppendorfs, added AHL into tubes, put back into shaker, as per protocol. Shake start again at 11:57
- Spun down cells, and added 500 μL of superntant to 1500 μL of T9002 cells at OD 0.1.
- Filled the 96 well plate, start shaking at 14:15
T9002 Testing
- OD of overnight culture = 1.23
- Recultured into 16 mL as per protocol, start shake at 9:42
- OD of second culture = 0.235
- Recultured into 25 mL, start shake at 12:25
J37016 Testing
- OD of overnight culture = 1.15
- Recultured into 16 mL as per protocol, start shake at 9:50
- OD of second culture = 0.247
- Recultured into 25 mL, start shake at 12:25
Ligations
- J37020 = J37032 (insert) -> J37031 (vector)
- Gene clean from gel pieces cut out yesterday
- Start ligation at 10:20
- Gene clean ligation at 12:30, ready for electroporation
==Aiia Western Blot== *Cultured a fresh day culture of S01656 (aiiA test) for a western blot at 9:30 *At 11.10 **Took a 1ml control **Added 4ul of 1molar IPTG to the remaining 4ml [western blot protocol] JohnChattaway 06:17, 22 August 2006 (EDT)
ImmunoTag PCR
- Successful PCR!!! All three repeats
- DNA run on gels and cut out and purified using the glass milk technique
- Ligation into T tail overhanging plasmid currently ongoing. Started at 2.50pm.
- 4.50pm- Ready for electroporation