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

{| width="800"
 * style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]] iGEM Project name 1
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

August 5th, 2009
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 -Extra digest to double check SpeI cuts 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 Will stop AscI/PacI digest at 3 hours 20 μL reaction DNA: 10 μL Buffer 4: 2 μL BSA: 2 μL AscI/PacI: .5 + .5 ddH2O: 5μL
 * 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
 * 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
 * Control (no enzyme), buffer 2
 * 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)
 * 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


 * }