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.
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