IGEM:IMPERIAL/2006/LabCalendar/2006-8-27

• IGEM:IMPERIAL/2006/LabCalendar/2006-8-27 Events
• Day in the lab
• Project Progress

To Do:

Miniprep (J27024 & J37018) and Maxiprep (J27024 & J37018 & 4G(S01656))

Ligation & Electroporation

• J37025 = B0034 + J37024
• J37022 = J04500 + J37024

Testing T9002, J37016, J37020

Culturing for Monday

• for testing T9002, J37016, J37020, J37022
• for Mini and Maxiprep J37025, J37022

JW: There was a problem on Saturday with the freezing of S01656, in that we did not realise until right at the end of the day that S01656 was Kanamycin resistant. We added ampicillin resistant LB and so the 10 Eppendorfs that are in the -80[[:Category:{{{1}}}|{{{1}}}]] freezer need to be thrown away. I cultured up some new S01656 so that someone could make up some new frozen stocks and this is in the shaker. The kanamycin is in an Eppendorf in the freezer.

Testing of T9002, J37016, J37020

• Took 16mL cultures out of shaker after 2h at 12:40
• OD measurements:
  • T9002 0.631
  • J37016 0.687
  • J37020 0.647
• Dilute down to OD 0.1 into 25mL
• Put 5mL tubes with AHL in shaker at 13:20 - to be taken out at 17:20

Miniprep of J37018 & J37024

• Miniprep successful
• PROBLEM: Gel did not run correctly - perhaps 1*TAE was not concentrated correctly, it might have been too concentrated...
• Miniprep has to be repeated to check the ligations for the above parts

Maxiprep of J37024, J37018, 4G

• Maxiprep successful

Ligations for J37022 and J37025

• Final Aiia construct: J37022 = 16P + J37024
• For Aiia predator cell: J37025 = 3O + J37024

• Digests of Maxiprep:
  • J37022:
    • Insert: J37024, cut with X & P (yellow buffer)
    • Vector: 16P. cut with S & P (yellow buffer!)
  • J37024:
    • Insert: J37024, cut with X & P (yellow buffer)
    • Vector: 3O, cut with S & P (yellow buffer!)
• Set up 30uL digest
• Leave in 37 C waterbath for 1h (13:00)
• Took out of waterbath at 13:55 and run digests on big gel
• PROBLEM: Gel did not run correctly - perhaps 1*TAE was not concentrated correctly, it might have been too concentrated...

• Shock! Horror! - we've realized that only one colony was cultured up for the minipreps today and a 'different' colony was cultured up for the maxiprep and from the gel we ran for the ligations we couldn't really decipher if part J37024 was there or not....so I guess we've lost a day and we'll have to spend tomorrow re-maxipreping and ligating etc.