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

Paraoxon Biosensor

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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