Imperial College/24 August 2008

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24 August 2008

Wet Lab

Cloning

Tuesday
  • Prepare plates growth characterisation later in week.
  • Prepare overnight cultures of the Terminator, Chlormaphemicol Acetyltransferase, mRFP - Terminator and GFP-3-mutB - Terminator in XL1-Blue for midiprepping on Wednesday.
  • Run trial PCR reactions for genomic template and/or pDR111 template PCR cloning steps and validate by fragment size on gel
Wednesday
  • Run PCR cloning reactions for those not done on Tuesday adn validate as before
Thursday
  • Cut PCR clones, ligate into Biobricks adn transform XL1-Blue E.coli
Friday
  • Trial single colony PCR method to validate correct insertion into Biobricks

B.subtilis

Tuesday
  • Prepare 1x10 ml LB in 100ml flask in the morning and inoculate with B.subtilis in the afternoon. This culture will be used for the microscope tomorrow.
  • Perform the B.subtilis transformation 1 , click here for the protocol, this time use 500ul of competent cells as opposed to 200ul as we have previously used.
Wednesday
  • Microscopy,
  • Check transformants,
  • Perform Colony PCR using on the transformed B.subtilis using primers to test for correct integration into the amyE site.
Thursday
  • Perform integration with linear DNA from the pDR110 vector by cutting with suitable enzymes. We will use the transformation protocol 1.
  • B.subtilis growth curve against O.D.600
Friday
  • If colonies have grown from thursday transformation then perform colony PCR on these colonies to check correct integration of linear DNA.