17 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
- 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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