IGEM:IMPERIAL/2006/LabCalendar/2006-8-22

• IGEM:IMPERIAL/2006/LabCalendar/2006-8-22 Events
• Day in the lab
• Project Progress

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

LoxP PCR

• Ran both PCR reactions today - joining the LoxP sites to B0021 (1M) and I13521 (18I)
• Used Template:25uL mix:
  • 15μL water
  • 2.5 μL Pfu turbo polymerase buffer
  • 1 μL 10 x diluted Primer 1 or 3
  • 5 μL 10 X diluted Primer 2 antisense or 2 sense
  • 0.5μL dNTPs
  • 0.5μL Pfu turbo polymerase
  • 0.5μL 10 x diluted maxiprep 18I or 1M

• Ran 3 PCR reactions with slightly different annealing temperatures for both of the reactions:
  • 1M - 38[[:Category:{{{1}}}|{{{1}}}]], 40 [[:Category:{{{1}}}|{{{1}}}]], 42[[:Category:{{{1}}}|{{{1}}}]]
  • 18I - 58 [[:Category:{{{1}}}|{{{1}}}]], 60{{c}, 62[[:Category:{{{1}}}|{{{1}}}]]

• Results
• The 18I PCR was not successful - will be run again tomorrow with slightly altered protocol
• The 1M PCR may have been successful - the gel was slightly clouded by the loading buffer and so will also be re-run to check results