Imperial College/24 August 2008

=24 August 2008=

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

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.