McGill/October
From 2007.igem.org
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Contents |
== October 2007 ==
October 1
Seeded E0434 and I5611 in 1X LB.
October 2
Diluted E0434 and I5611 a total of 15X in 1X LB.
4.5ml LB (1x)
300ul culture
E0434 - 5ul Amp/Kan, I5611 - 5ul Amp
October 3
Did the 15X dilution again as the total volume was not enough to perform a midiprep...stupid me! (Avi).
- Want 200ml of dilution in total in order to allow the culture to grow efficiently overnight.
E0434
100ml, 100ml LB 2X, 1.5ml Kan, 200ul Amp, 2ml culture
I5611
100ml, 100ml LB 2X, 200ul Amp, 2ml culture
October 5
Jimmy and I midiprepped 50mL of each E0434 & I5611 using Annette's procedure. Could not prep more because 1. annette's procedure required the clinical centrifugue where there are only 6 spots for 15ml tubes, 2.there were not enough sterile epi tubes, 3. could not use real Qiagen procedure instead (the rotor we needed in the big centrifuge is broken). I'm not sure about the yield of this. definitely check the dna conc before screening. one of the last steps in annette's procedure involving the pooling of pellets of DNA from 6 epi tubes to 1 tube using ethanol is iffy and there may be a lot of dna loss there.
I've lysated the pellet from another 50mL of each (w/P1) and froze it in case we want to do this again. It has green tape around it. The 2 epi tubes w/ the midi are next to it and also have green tape around them, in the top right corner of the freezer. Horia
October 9
Screening digest of Horia's midiprep:
E0434
1) Xba + SpeI - cuts at 3181bp & 925 bp
2) HindIII + PstI - cuts at 2.8kb & 1.3kb
3) EcoRI - 4.1kb
I5611
1) Xba + PstII - cuts at 4.2kb & 2.1kb
2) EcoRI + BsrGI - cuts at 4.1kb & 2.2kb
3) EcoRI - 6.3kb
October 10
Screen again as gel had too much EtBr, so bands could not be seen.
October 11
Screening was done again, this time it was concluded that there was no DNA in the midiprep, so the midiprep was done incorrectly, or no DNA was salvaged. Ahhh! Taking a break from this cursed lab and waiting for that darned I-brick to show up!
