IGEM:University of Chicago/2009/Notebook/Paraoxon Biosensor/2009/08/04

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August 4th, 2009
20μL reaction: + DNA: RFP/mCherry GFP-Kan NEB Buffer 4              2μL            2μL BSA (10x)                 2μL            2μL ddH2O                     10μL           10μL Enzymes(AscI, PacI)     .5 + .5         .5 + .5 DNA                       5μL            3μL 20 μL reaction: AscI     PacI      AscI/PacI NEB Buffer       1μL       1μL       1μL BSA               -        1μL       1μL Enzyme          .5μL      .5μL      .5 + .5 DNA              1μL       1μL       1μL ddH2O           7.5μL     6.5μL     6μL
 * Digest and ligation!
 * PCR purify yesterday's product using Qiagen kit
 * restriction digest:
 * Ran at 37°C for 1 hr
 * heat inactivate at 65°C for 20 minutes
 * gel purify
 * Run on 1.2% agarose gel - 20μL reaction run
 * Ligation:
 * 2-3x molar ratio of insert to vector
 * 20 μL reaction
 * room temperature for 1 hour
 * Problem: Restriction digest looks like it didn't work
 * Only 1 band seen on AscI/PacI double digest (after cutting out fragments)
 * Need to see if enzymes work: New digest!
 * Digest: PacI, AscI, PacI/AscI
 * 1 hour at 37°C
 * run on 1.2% agarose gel
 * Gel will be posted here
 * Notes: Almost looks like AscI is working (fewer super colored bands) bu PacI isn't
 * Will try new combination of digests tomorrow


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