IGEM:University of Chicago/2009/Notebook/Paraoxon Biosensor/2009/08/05

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August 5th, 2009

  • Possible source of problem: Satoe and Chichi recommend larger reaction volume because glycerol from restriction enzyme stock can inhibit reaction
  • larger reaction volume (30μL+) also recommended to have smaller template DNA concentration


  • Concentration of DNA stock (made on 6/21/09) is 426 μg/mL or .46ng/μL
  • Concentration of RFP: 5.56 μg/mL or 5.5 ng/μL
  • Concentration of mCherry: 6.6 μg/ml or .46 ng/μL
Digest:
               50μL reaction      mCherry      RFP         Control
DNA               1 μL             5 μL        5 μL          1μL
                 (plasmid)        (insert)    (insert)     (GFP-Kan)
NEB Buffer 4      1 μL             5 μL        5 μL          5μL
10x BSA           5 μL             5 μL        5 μL          5μL
Enzyme          .25 + .25       .25 + .25    .25 + .25     .25μL PacI
ddH2O            38.5μL           34.5μL      34.5μL        39μL
Total             50μL             50μL        50μL         50μL
  • Run for 3 hours
  • Note: .25μL of enzyme should digest 2.5 μg of DNA, so we have a 5-fold excess of enzyme for plasmid, 50-fold for insert
  • Gel here!
  • Not much RFP/mcherry visible
  • Gel Purification necessary?


  • Digest possibilities:
  • PacI/BglII -> Buffer 2 and BSA at 37°C, extra enzyme
  • AscI/SpeI -> Buffer 4 and BSA at 37°C
  • SpeI/BglII -> Buffer 2 and BSA at 37°C, extra enzyme

-Extra digest to double check SpeI cuts

  • Control (no enzyme), buffer 2
Digest:

             PacI/BglII     AscI/SpeI     SpeI/BglII    Control
NEB Buffer    2, 1μL         4, 1μL        2, 1μL        2, 1μL
BSA            1 μL           1 μL          1 μL          1 μL
Enzyme       .75 + .75       .5 + .5      .75 + .75        -
ddH2O          5.5μL          6 μL          5.5 μL        7 μL
DNA            1 μL           1 μL          1 μL          1 μL

Total: 10 μL reactions
  • Ran restriction digest
  • Gel inserted here
  • 3 hour digest worked! Despite low volume and high enzyme concntration
  • PacI and AscI both work (fragment size is correct)

Will stop AscI/PacI digest at 3 hours

  • Also tried gel purification of mcherry/RFP


  • Gel purification
  • Added 600μL ADB buffer
  • elute in 20μL elution buffer, incubate for 1 minute then spen down
  • check on gel to confirm purity, use leftover digest material as control
  • take concentration


  • Restriction digest
20 μL reaction
DNA: 10 μL
Buffer 4: 2 μL
BSA: 2 μL
AscI/PacI: .5 + .5
ddH2O: 5μL