Imperial College/17 August 2008

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Revision as of 15:50, 5 September 2008

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17th August 2008

Wet Lab

B.subtilis

Monday
  • Prepare media and reagents for the protocol transformation 1 protocol
  • Prepare 2x10ml LB media in 100ml flasks
  • Prepare 2x100ml LB media in 1000ml flasks
  • Prepare 2x overnight cultures in the 10ml LB media flasks
  • Prepare a 1% agarose gel and run the digest prepared on friday
Tuesday
  • Prepare competent cells using the transformation 2 protocol, this time growing cells to an o.D.600
  • Prepare 1x20ml of LB media in a 200ml flask (autoclaved in the morning)
  • Prepare 1x20ml of SpC media in a 200ml flask (autoclaved in the morning)
  • Prepare aliquots of SpII media (autoclaved in the morning)
Wednesday
  • Prepare competent cells using the transformation 1 protocol,
  • Electroporate the compete cells from the transformation protocol 2 using a range of concentrations
Thursday
  • Transform the competent cells prepared from transformation 2 protocol
  • Check the transformation cells from protocol 2, if transformed correctly then carry the test for correct integration,
Friday
  • If the transformation has been successful then carry out the integration test.

Cloning

Monday
  • Prepare plates and Media for later in the week
  • Transform E.coli Xl1-blue with C0012 (LacI) from the registry again
Tuesday
  • Check LacI transformants
  • Miniprep and digest 0.1.01 (Double Terminator) and 0.1.02 (Chloraphenicol Reisitance).
    • Ligate together to form construct 0.1.14 and transform into Xl1-Blue E.coli
Wednesday
  • Check E.coli transformed with construct 0.1.14 for growth and by PCR (if growth was succesful)
  • If primers have arrived, PCR clone LacI, XylR, Spectinomycin, EpsE modified integration sequnce and AmyE integration sequence into Biobricks
Thursday
  • If grown, mini/midi-prep a few colonies from each plate and digest a sample with XbaI and SpeI to determine which plasmids have the insert in the correct orientation.
  • If primers arrive today PCR cloning should be carried out
Friday
  • If primers arrived Thursday check plates for growth and carry out minipreps
  • PCR check the minipreps that could be digested by XbaI and SpeI

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