IGEM:University of Chicago/2009/Notebook/Paraoxon Biosensor/2009/06/29

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June 29, 2009

  • Primers have arrived! Hurrah for Invitrogen!
  • Primers come lyophilized. Need to dilute in sterile TE buffer to 100μM solution. Time for some math....
YGR035Cdel-F => 8.58nmol ==> add 85.8μL
YGR035Cdel-R => 17.42nmol ==> add 174.2μL
YHR139Cdel-F => 18.26nmol ==> add 186.2μL
YHR139Cdel-R => 15.01 nmol ==> add 150.1 μL
YOR049Cdel-F => 17.68nmol ==> add 176.8μL
YOR049Cdel-R => 19.04 nmol ==> add 190.0 μL
YOR186Wdel-F => 14.49nmol ==> add 149.5 μL
YOR186Wdel-R => 10.33nmol ==> add 103.3 μL
YGR213Cdel-F => 8.45nmol ==> add 85.5μL
YGR213Cdel-R => 17.93nmol ==> add 179.3μL
YGR236Cdel-F => 7.79nmol ==> add 77.9μL
YGR236Cdel-R => 17.02 nmol ==> add 170.0μL
YLR346Cdel-F => 18.65nmol ==> add 186.5μL
YLR346Cdel-R => 15.08 nmol ==> add 150.1μL
  • Add TE, incubate at room temperature for .5-3 hours
  • Then dilute each to 5μM for PCR

PCR

  • Use Accusure (supplied by Satoe)
  • More accurate polymerase than Taq
PCR program
1. Incubate: 95C 10min
2.-         95C 30sec
3. annealing: 58C 30sec
4. elongation: 68C 4min
5. Store:      12C --
  • Run steps 2--4 30 times
  • PCR recipe (20μL)
  • Note from 7/1/09: THIS RECIPE IS WRONG. Real recipe from Satoe was misinterpreted (by Nora who is very, very sorry) in this fashion until 7/1. For real recipe please refer to protocols. In retrospect this recipe seems totally crazy, but at least we've learned our lesson. :)
10X buffer:4μL
dNTP's: 1μL
Template (using TL1): .5μL
Primer 1: 4μL
Primer 2: 4μL
Polymerase: 2μL  (*cough* *cough*)
ddH2O: 2μL
Total: 20μL
  • Run overnight, pick up in the morning and run gel