IGEM:IMPERIAL/2006/LabCalendar/2006-8-28

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Contents

To Do:

*Repeat Mini and Maxiprep of J37024 (colonies a-e) & J37018 (colonies a-d)
 !!!IMPORTANT NOTE!!!: Use 1.5mL from the 100mL cultured colony for maxiprep and store 0.5mL of each colony in fridge 
                       for any further culturing
                       Also, any cells containing final parts should be put into -80 C freezer on that day
*Repeat Ligation & Electroporation: 
      - J370025 = B0034 + J37024
      - J37022 = J04500 + J37024
*Electroporation of J37015
*Make frozen stock of cells of S01656 (Kanamycin) and J37018
*Testing T9002, J37016, J37020
*Culturing for Tuesday: for testing T9002, J37016, J37020

T9002/J37016/J37020

  • T9002 OD600 after o/n culture: 2.0
  • T9002 OD600 after o/n culture: 2.0
  • T9002 OD600 after o/n culture: 2.0
  • Testing conducted according to protocol.
  • 5mL tubes taken out at 17:20 and transferred into 96 well plate
  • Start reading at 18:40 - 20:30 (proceeded with ligations in between)

Minipreps

  • J37018: All four colonies that were minipreped showed the same results but somebody should double check the picture - I can't see the expected bands.
  • J37024: same as above.

Maxipreps

  • J37024 (a,c & d) were successful. Maxiprep of colony b may be dubious.

Ligations

  • Colony c of J37024 was cut out of the gel from the maxiprep digest in order to be ligated (GEL HAS TO BE DOUBLE CHECKED...)
  • Gel for 3O and 16P had expected band sizes
  • Glass Milk purification
  • Ligation was set up at 18:00 (leave for at least one hour)
  • Left for 1.5h to ligate, following that, then 2nd time glass milk purification (modified version)
  • Successful electroporation - could not electroporate J37015 because NO COMPETENT CELLS LEFT! (there were only 2 left)
  • Plates with the 2 ligated parts put in incubater overnight (21:00)

Creating a Frozen Stock of Cells

  • Has to be done tomorrow.

For Tomorrow

  • Cultured T9002/J37016/J37020 from frozen stock

General Things to do

  • We need to make up more 50 times TAE (we've used up a lot this weekend for the ligations. Also, there was something wrong with the old batch of TAE. For gels in the near future, use the 1 times TAE that has been dated 27/8/06 or 28/8/06.)
  • We also need more LB and autoclaved flasks and cuvettes. Do we need more plates?
  • There are about 10 LB Amp plates left in the cold room
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