IGEM:University of Chicago/2009/Notebook/Organophosphate Hydrolase/2009/07/09

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Goal: Need to miniprep, quantify, digest/agarose pCTCON2-GFP from...

11:00 - Started 4 hour EBY100 cultures for transfection from single colony tubes 3 x 10 ml, OD660 .203, .166, .194.

Aliquotted 12 rxns worth of Zyppy miniprep kits. Annie miniprepped pCTCON2-GFP plasmid (30 ul TE@165.4 ug/ml)

Restriction digest to confirm: BAMHI and EcoRI, 1 hour @ 37 degrees C

1 ul 10 x buffer

1ul 10 x BSA

0.5 ul EcoRI

0.5 ul BamHI

1 ul DNA

6 ul H2O

14:40 start incubation

-Insert image here-

Transformation of pCTCON2-GFP into EBY100

                         Final OD660: 0.428

Resuspended twice in LiOAc b/c first did it in 500 ml. Heatshock 25 min instead of 20 min. Used herring sperm ssDNA b/c Salmon sperm ssDNA thick like jelly.

Provided by Eric: /[A-Z] 6898/? Used 3 ul b/c 5 mg/ml instead of 10

Plated control, pCTCON2- (?), pCTCON2-GFP, 8 ul each.

DNAwt: N/A, 9.6 ug(!?), 1.28 ug

Tomorrow:

  Figure out mystery tubes.
  Repeat restriction digest? (or at least figure out expected fragments)
  Make glycerol stocks of EBy100; E. coli w/ pCTCON2,E.coli w/pCTCON2-GFP
  Saturday: check today's transformants
  check Nora's transformants from yesterday
  design paraoxon trials