Imperial College/17 August 2008

=17 August 2008=

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.

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