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

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August 4th, 2009

  • Digest and ligation!

  • PCR purify yesterday's product using Qiagen kit

  • restriction digest:
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
  • 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
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
  • 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