IGEM:IMPERIAL/2006/LabCalendar/2006-9-1

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TO DO:
*Testing of T9002, J37016, J37020, S01656(contd. from Thursday), J37015, J37015RS
*Mini & Maxiprep of new J37024 (CHECK the miniprep gel straight after !)
*Ligation & electroporation of J37022, J37025 (using new J37024)
*Finish biosensor protocol from Thursday (read fluorescence)

Contents

Testing T9002/J37016/J37020

  • OD of o/n culture of T9002: 2.00
  • OD of o/n culture of J37016: 2.00
  • OD of o/n culture of J37020: 2.00
  • Put in the shaker at 9.25. Take out at 11.25.

After 2 hrs:

  • Took OD of cultures, innoculated into 25ml, placed in shaker at 11:45. To be taken out at 3:45.

Miniprep of J37024 to check ligations

Maxiprep

  • Maxiprep 3 samples of J37024 II
  • Throw away original maxiprep of J37024 (I) as minipreps show it to have not been successful

Ligations

  • All three samples of J37024 II inserted into vectors 3O Predator (AiiA contstruct for predator cell J37025) and 16P (AiiA test construct J37022)

LoxP PCR

  • We altered the annealing temperatures once more in a final attempt to get this PCR to work

5 cycles at 37[[:Category:{{{1}}}|{{{1}}}]]
5 cycles at 58[[:Category:{{{1}}}|{{{1}}}]]
20 cycles at 68[[:Category:{{{1}}}|{{{1}}}]]

  • It seems that it may have at last worked because we have a band at the expected size (~1kb)
  • However this band is smeared so we cannot be sure of its success - but it certainly is promising nonetheless
  • We will now attempt the PCR fusion on Monday (04/09), which hopefully will give us the completed construct.

Rosetta competent cells

  • We were only able to get 3 colonies from the BL21 competent cells that were transformed yesterday. Therefore it was decided that we should try another type of cell as well.
  • The Rosetta competent cells were electroporated with S01656, and will be cultured up on Sunday in order that we can perform a Western blot on Monday.
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