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)

LoxP PCR

• Ran both PCR reactions today - joining the LoxP sites to B0021 (1M) and I13521 (18I)

• We are using Pfu turbo for these PCRs because we do not want an AT to be added by the polymerase to the end of the PCR product. This is what is done by Taq polymerase

• Used 25μL mix:
  • 15μL water
  • 2.5μL Pfu turbo polymerase buffer
  • 1μL 10 x diluted Primer 1 or 3
  • 5μL 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[[:Category:{{{1}}}|{{{1}}}]], 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

• One potential problem is the low annealing temperature required for one of the primers - around 40[[:Category:{{{1}}}|{{{1}}}]]
• If it does not work, then we have the possibility to shorten the restriction site and lengthen the annealing site, hence increasing the annealing temperature.

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

J37015 Positive Feedback Loop

• The OD600 of the o/n culture was measured
• Culture was then innoculated into 2ml of of fresh LB amp to get down to OD600 of 0.1
• Fresh culture left in shaker for 2hrs
• After two hours took OD and innoculated again into 2ml of LB amp to get OD600 of 0.1
• The above was repeated throughout the day (at 9.30, 11.45, 2.00, 4.15, 6.15)
• Final culture (at 6.15) stored in fridge for J37015 testing tomorrow. Hopefully the dilution method will work:-)