Difference between revisions of "Team:Northwestern/Lab"

 
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InterLab Experiment
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Project: NU iGEM 2017 Shared Project
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<html>
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Authors: Karen Taylor
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<title>Northwestern Template</title>
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Dates: 2017-06-26 to 2017-07-13
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<meta charset="UTF-8">
  
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Monday, 6/26
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<meta name="viewport" content="width=device-width, initial-scale=1">
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Goal of InterLab Experiment: Establish a GFP measurement protocol based on engineering principles that anyone with a plate reader can use in their lab. Teams will use the same exact protocol around the world to produce common, comparable units for measuring GFP with different plate readers.
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<link rel="stylesheet" href="https://www.w3schools.com/w3css/4/w3.css">
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<link rel="stylesheet" href="https://cdhttps://2017.igem.org/njs.cloudflare.com/ajax/libs/font-awesome/4.7.0/css/font-awesome.min.css">
  
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Friday, 7/7
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<head>
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Goal: Perform Parts 1 and 2 of the Interlab - Calibration measurements
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<script>
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Calibration measurements (Parts 1 and 2)
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The InterLab Plate reader Protocol was followed.
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Modifications:
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The plate was covered using aluminum foil prior to avoid contact with light prior to placing in the plate reader
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The plate reader took measurements 3 times and the results were averaged
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Raw Data
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A
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B
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C
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D
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E
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1
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1 2 3 4
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2
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LDX - HS40 100% 0.043 0.047 0.045 0.046
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3
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0.043 0.047 0.045 0.047
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4
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0.043 0.047 0.045 0.046
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5
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H2O 0.036 0.036 0.036 0.036
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6
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0.036 0.035 0.036 0.036
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7
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0.036 0.036 0.036 0.036
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Raw Data - InterLab Part 1
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A
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B
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C
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D
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E
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F
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G
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H
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I
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J
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K
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L
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M
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1
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1 2 3 4 5 6 7 8 9 10 11 12
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2
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A OVRFLW OVRFLW OVRFLW OVRFLW 83331 47636 25670 13596 7061 3708 2036 246
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3
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OVRFLW OVRFLW OVRFLW OVRFLW 81361 46611 25090 13282 6921 3636 1997 242
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4
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OVRFLW OVRFLW OVRFLW OVRFLW 81215 46394 25044 13246 6903 3630 1990 240
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5
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B OVRFLW OVRFLW OVRFLW OVRFLW 84505 47496 26029 13769 7133 3734 1995 247
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6
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OVRFLW OVRFLW OVRFLW OVRFLW 82862 46634 25546 13507 7008 3663 1958 244
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7
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OVRFLW OVRFLW OVRFLW OVRFLW 82738 46476 25452 13473 6990 3655 1956 242
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8
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C OVRFLW OVRFLW OVRFLW OVRFLW 84474 47497 25109 13643 6942 3649 2077 256
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9
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OVRFLW OVRFLW OVRFLW OVRFLW 83244 46778 24730 13444 6831 3591 2050 253
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10
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OVRFLW OVRFLW OVRFLW OVRFLW 83057 46662 24663 13405 6819 3586 2052 254
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11
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D OVRFLW OVRFLW OVRFLW OVRFLW 78379 40604 23035 11854 6424 3178 1839 246
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12
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OVRFLW OVRFLW OVRFLW OVRFLW 77456 40112 22778 11704 6332 3129 1814 244
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13
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OVRFLW OVRFLW OVRFLW OVRFLW 77326 40069 22742 11686 6324 3115 1807 244
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Raw Data - InterLab Part 2
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Emailed Jake from iGEM regarding OVERFLOW: need to change sensitivity and redo this part
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Data Analysis
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The data was analysed by following instructions found in the InterLab protocol.
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A
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B
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C
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1
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Microplate Reader Measurements
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2
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H2O LDX-HSO4 100%
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3
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Replicate 1 0.036 0.043
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4
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Replicate 2 0.0356666 0.047
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5
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Replicate 3 0.036 0.045
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6
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Replicate 4 0.036 0.0463333
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7
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Average 0.03591665 0.045333325
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8
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Corrected Abs600 0.009416675
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9
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Reference OD600 0.0425
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10
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Correction factor 0.2215688235
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Microplate Reader Data - InterLab Part 1
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Tuesday, 7/11
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$(document).ready(function () {
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Transformations
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    var parentDivs = $('#nestedAccordion div'),
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Goal: Complete transformations of 6 Testing Devices and 2 controls
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        childDivs = $('#nestedAccordion h3').siblings('div');
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Materials
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Resuspended DNA to be transformed
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○
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Resuspend DNA Distribution Kit wells with 10uL dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye
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10pg/µl Positive transformation control DNA
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Competent Cells (50µl per sample)
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2ml Microtubes
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One tube per transformation
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Label tubes with part name or well location before starting
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SOC Media (200µL per sample)
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Petri plates w/ LB agar and antibiotic (2 per sample)
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2 plates per transformation
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CAM
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Equipment
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Floating Foam Tube Rack
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Ice & ice bucket
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Fill bucket, pre-chill 2mL tubes for 5 min
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Thaw competent cell stock on ice for 10-15 min
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Lab Timer
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42°C water bath
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37°C incubator
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Sterile spreader or glass beads
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Pipettes and Tips (10µl, 20µl, 200µl tips and pipettes recommended)
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Transformation Protocol
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Resuspend DNA in selected wells in the Distribution Kit. Label 2ml tubes with part name or well location. Fill lab ice bucket with ice, and pre-chill 2ml tubes (one tube for each transformation, including your control) in a floating foam tube rack.
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1.
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Thaw competent cells on ice: This may take 10-15min for a 260µl stock. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.
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2.
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Pipette 50µl of competent cells into 2ml tube: 50µl in a 2ml tube per transformation. Tubes should be labeled, pre-chilled, and in a floating tube rack for support. Keep all tubes on ice. Don’t forget a 2ml tube for your control.
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3.
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Pipette 1µl of resuspended DNA into 2ml tube: Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice.
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4.
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Pipette 1µl of control DNA into 2ml tube: Pipette 1µl of 10pg/µl control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.
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5.
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Close 2ml tubes, incubate on ice for 30min: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
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6.
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Heat shock tubes at 42°C for 1 min: 2ml tubes should be in a floating foam tube rack. Place in water bath to ensure the bottoms of the tubes are submerged. Timing is critical.
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7.
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Incubate on ice for 5min: Return transformation tubes to ice bucket.
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8.
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Pipette 200µl SOC media to each transformation: SOC should be stored at 4°C, but can be warmed to room temperature before use. Check for contamination.
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9.
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Incubate at 37°C for 2 hours, shaker or rotor recommended:
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10.
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Pipette each transformation on two petri plates for a 20µl and 200µl plating: Pipette 20µl and 200µl of the transformation onto appropriately labeled plates. Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
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11.
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Incubate transformations overnight (14-18hr) at 37°C: Incubate the plates upside down (agar side facing up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.
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Wednesday, 7/12
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    $('#nestedAccordion button').click(function () {
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The 200μL plates were selected for the overnight culture since more colonies were present.
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        parentDivs.slideUp();
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InterLab Plates (1/2)
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        if ($(this).next().is(':hidden')) {
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            $(this).next().slideDown();
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        } else {
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            $(this).next().slideUp();
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        }
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    });
  
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thumbnail
 
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InterLab Plates (2/2)
 
  
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thumbnail
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    $('#nestedAccordion h3').click(function () {
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Kit plate 6:
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        childDivs.slideUp();
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B (positive control) I20270
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        if ($(this).next().is(':hidden')) {
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D (negative control) R0040
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            $(this).next().slideDown();
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F Test Device 1 J364000
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        } else {
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H Test Device 2 J364001
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            $(this).next().slideUp();
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J Test Device 3 J364002
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        }
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L Test Device 4 J364003
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    });
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N Test Device 5 J364004
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});
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P Test Device 6 J364005
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Plasmids have pSBIC3 plasmid backbone
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Purpose: Create overnight cultures from the plated bacteria.
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Procedure:
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Pick 2 colonies from each of plate and inoculate it on 5-10 mL LB medium + Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm - STEP COMPLETE
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Note: 5mL of LB were added
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Thursday, 7/13
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</script>
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Purpose: Cell growth, sampling and assay
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◻ Set your instrument to read OD600 (as OD calibration setting)
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◻ Measure OD600 of the overnight cultures
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◻ Record data in your notebook
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◻ Import data into Excel ( Dilution Calculation ) Sheet_1 provided
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◻ Dilute the cultures to a target OD 600 of 0.02 (see the volume of preloading culture and media in Excel ( Dilution Calculation ) Sheet_1) in 12 m l LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light).
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◻ Incubate the cultures at 37°C and 220 rpm.
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◻ Take 500 μL samples of the cultures at 0, 2, 4, and 6 hours of incubation. (At each time point, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 samples per time point)
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◻ Place samples on ice.
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◻ At the end of sampling point you need to measure your samples (OD and Fl measurement), see the below for details.
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◻ Record data in your notebook
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◻ Import data into Excel ( cell measurement tab ) Sheet_1 provided
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- Calibration measurements were redone on 7/13 to account for overflow. Instrument sensitivity was changed
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- A gain of 35 degrees was used.
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Calibration curve:
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Standard Curve
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thumbnail
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</head>
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iGEM Sheet to record all measurements:
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Northwestern_Interlab_Dilution_Calculation_Sheet (1).xlsx
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<body>
  
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Northwestern_University_InterLab_2017_Measurements.xlsx
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<br>
  
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All measurements are complete and forms have been submitted to iGEM (Saturday July 15th 2017)
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<center><h1 style="padding-top:2%; padding-right: 15%; padding-left:15%"><br><br>Lab Notebook</h3></center>
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INTERLAB COMPLETE
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<br>
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<div id="nestedAccordion" class="w3-center">
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  <button class="w3-button w3-block panel-collapse collapse" style="font-size:30px; padding-top: 2%; padding-bottom: 2%"> Weekly summaries</button>
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    <div style="display:none;">
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        <h3 class="w3-button w3-block"> Week 1 and 2</h3>
 +
        <div> <center><object data="https://static.igem.org/mediawiki/2017/archive/3/3d/20170802184027%21T--Northwestern--Week2.pdf" type="application/pdf" width="75%" height="600px">
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Your device does not support embed PDFs. Please click the following link to open up the PDF. <a href="https://static.igem.org/mediawiki/2017/archive/3/3d/20170802184027%21T--Northwestern--Week2.pdf"></a>
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</object> </center> </div>
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        <h3 class="w3-button w3-block"> Week 3</h3 >
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        <div> <center><object data="https://static.igem.org/mediawiki/2017/d/d9/T--Northwestern--Week3.pdf" type="application/pdf" width="75%" height="600px">
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Your device does not support embed PDFs. Please click the following link to open up the PDF. <a href="https://static.igem.org/mediawiki/2017/d/d9/T--Northwestern--Week3.pdf"></a>
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</object> </center> </div>
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        <h3 class="w3-button w3-block"> Week 4</h3 >
 +
        <div> <center><object data="https://static.igem.org/mediawiki/2017/6/66/T--Northwestern--Week4.pdf" type="application/pdf" width="75%" height="600px">
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Your device does not support embed PDFs. Please click the following link to open up the PDF. <a href="https://static.igem.org/mediawiki/2017/6/66/T--Northwestern--Week4.pdf"></a>
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</object> </center> </div>
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          <h3 class="w3-button w3-block"> Week 5</h3 >
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        <div>  <center><object data="https://static.igem.org/mediawiki/2017/1/16/Lab_Notebook_-_Week_5_%287-10-17-7-16-17%29_2017-07-10_-_2017-07-14_%28etr_XTi9rICh%29.pdf" type="application/pdf" width="75%" height="600px">
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</object> </center> </div>
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          <h3 class="w3-button w3-block"> Week 6</h3 >
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        <div> <center><object data="https://static.igem.org/mediawiki/2017/e/ee/T--Northwestern--Week6.pdf" type="application/pdf" width="75%" height="600px">
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Your device does not support embed PDFs. Please click the following link to open up the PDF. <a href="https://static.igem.org/mediawiki/2017/e/ee/T--Northwestern--Week6.pdf"></a>
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</object> </center> </div>
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          <h3 class="w3-button w3-block"> Week 7</h3 >
 +
        <div> <center><object data="https://static.igem.org/mediawiki/2017/b/b9/T--Northwestern--Week7.pdf" type="application/pdf" width="75%" height="600px">
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Your device does not support embed PDFs. Please click the following link to open up the PDF. <a href="https://static.igem.org/mediawiki/2017/b/b9/T--Northwestern--Week7.pdf"></a>
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</object> </center> </div>
 +
        <h3 class="w3-button w3-block"> Week 8-9</h3 >
 +
        <div> <center><object data="https://static.igem.org/mediawiki/2017/7/72/T-Northwestern--Week8.pdf" type="application/pdf" width="75%" height="600px">
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Your device does not support embed PDFs. Please click the following link to open up the PDF. <a href="https://static.igem.org/mediawiki/2017/7/72/T-Northwestern--Week8.pdf"></a>
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</object> </center> </div>
 +
 
 +
        <h3 class="w3-button w3-block"> Week 10</h3 >
 +
        <div> <center><object data="https://static.igem.org/mediawiki/2017/d/d7/T-Northwestern--Week10.pdf" type="application/pdf" width="75%" height="600px">
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Your device does not support embed PDFs. Please click the following link to open up the PDF. <a href="https://static.igem.org/mediawiki/2017/d/d7/T-Northwestern--Week10.pdf"></a>
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</object> </center> </div>
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 +
    </div>
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 +
<button class="w3-button w3-block" style="font-size : 30px; padding-top: 2%; padding-bottom: 2%"> Protocols  </button>
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    <div style="display:none;">
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    <h3 id = "input" class="w3-button w3-block"> Making TAE buffer</h3 > 
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<div> <center><object data="https://static.igem.org/mediawiki/2017/c/ca/T--Northwestern--10xTAE.pdf
 +
" type="application/pdf" width="75%" height="600px">
 +
Your device does not support embed PDFs. Please click the following link to open up the PDF. <a href="https://static.igem.org/mediawiki/2017/c/ca/T--Northwestern--10xTAE.pdf
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"></a>
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</object> </center> </div>
 +
 
 +
    <h3 class="w3-button w3-block"> Culture Innoculation</h3 >
 +
      <div>  <center><object data="https://static.igem.org/mediawiki/2017/7/72/T--Northwestern--CultureInoculation.pdf
 +
" type="application/pdf" width="75%" height="600px">
 +
Your device does not support embed PDFs. Please click the following link to open up the PDF. <a href="https://static.igem.org/mediawiki/2017/7/72/T--Northwestern--CultureInoculation.pdf
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"></a>
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</object> </center> </div>
 +
 
 +
    <h3 class="w3-button w3-block"> Gemstone dye protocol</h3 >
 +
      <div> <center><object data="https://static.igem.org/mediawiki/2017/f/f7/T--Northwestern--GemstoneDye.pdf
 +
" type="application/pdf" width="75%" height="600px">
 +
Your device does not support embed PDFs. Please click the following link to open up the PDF. <a href="https://static.igem.org/mediawiki/2017/f/f7/T--Northwestern--GemstoneDye.pdf
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"></a>
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</object> </center> </div>
 +
 
 +
 
 +
      <h3 class="w3-button w3-block"> Fractionation</h3 >
 +
      <div> <center><object data="https://static.igem.org/mediawiki/2017/1/18/T--Northwestern--Fractionation.pdf
 +
" type="application/pdf" width="75%" height="600px">
 +
Your device does not support embed PDFs. Please click the following link to open up the PDF. <a href="https://static.igem.org/mediawiki/2017/1/18/T--Northwestern--Fractionation.pdf
 +
"></a>
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</object> </center>  </div>
 +
   
 +
      <h3 class="w3-button w3-block"> LB + Plate</h3 >
 +
      <div> <center><object data="https://static.igem.org/mediawiki/2017/d/da/T--Northwestern--LBPlate.pdf
 +
" type="application/pdf" width="75%" height="600px">
 +
Your device does not support embed PDFs. Please click the following link to open up the PDF. <a href="https://static.igem.org/mediawiki/2017/d/da/T--Northwestern--LBPlate.pdf
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"></a>
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</object> </center> </div>
 +
 
 +
      <h3 class="w3-button w3-block"> Supplemented Media</h3 >
 +
        <div> <center><object data="https://static.igem.org/mediawiki/2017/7/73/T--Northwestern--M9SupplementedMedia.pdf
 +
" type="application/pdf" width="75%" height="600px">
 +
Your device does not support embed PDFs. Please click the following link to open up the PDF. <a href="https://static.igem.org/mediawiki/2017/7/73/T--Northwestern--M9SupplementedMedia.pdf
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"></a>
 +
</object> </center> </div>
 +
       
 +
        <h3 class="w3-button w3-block"> Transformations</h3 >
 +
        <div> <center><object data="https://static.igem.org/mediawiki/2017/f/f8/T--Northwestern--TransformationProtocol.pdf
 +
" type="application/pdf" width="75%" height="600px">
 +
Your device does not support embed PDFs. Please click the following link to open up the PDF. <a href="https://static.igem.org/mediawiki/2017/f/f8/T--Northwestern--TransformationProtocol.pdf
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"></a>
 +
</object> </center> </div>
 +
       
 +
          <h3 class="w3-button w3-block"> Miniprep (Promega)</h3 >
 +
          <div> <center><object data="https://static.igem.org/mediawiki/2017/9/9b/T--Northwestern--MiniprepPromega.pdf
 +
" type="application/pdf" width="75%" height="600px">
 +
Your device does not support embed PDFs. Please click the following link to open up the PDF. <a href="https://static.igem.org/mediawiki/2017/9/9b/T--Northwestern--MiniprepPromega.pdf
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"></a>
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</object> </center> </div>
 +
       
 +
          <h3 class="w3-button w3-block"> OMV Purification</h3 >
 +
          <div> <center><object data="https://static.igem.org/mediawiki/2017/e/e0/T--Northwestern--OMVPurification.pdf
 +
" type="application/pdf" width="75%" height="600px">
 +
Your device does not support embed PDFs. Please click the following link to open up the PDF. <a href="https://static.igem.org/mediawiki/2017/e/e0/T--Northwestern--OMVPurification.pdf
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"></a>
 +
</object> </center> </div>
 +
<h3 class="w3-button w3-block">PCR for Removal of His6 Tag from DsbA</h3 >
 +
            <div> <center><object data="https://static.igem.org/mediawiki/2017/9/91/T--Northwestern--PCRHisRemoval.pdf
 +
" type="application/pdf" width="75%" height="600px">
 +
Your device does not support embed PDFs. Please click the following link to open up the PDF. <a href="https://static.igem.org/mediawiki/2017/9/91/T--Northwestern--PCRHisRemoval.pdf
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"></a>
 +
</object> </center> </div>
 +
 
 +
            <h3 class="w3-button w3-block">Sequencing</h3 >
 +
            <div> <center><object data="https://static.igem.org/mediawiki/2017/7/71/T--Northwestern--Sequencing.pdf
 +
" type="application/pdf" width="75%" height="600px">
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Latest revision as of 03:55, 2 November 2017

Northwestern Template Northwestern Template

Overview Silver Gold Collaborations Engagement
Overview
Experiments Modeling Results Notebook Safety Interlab
Description Design Medal Criteria
About Us Attributions

Northwestern Template



Lab Notebook


Week 1 and 2

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Week 3

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Week 4

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Week 5

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Week 6

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Week 7

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Week 8-9

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Week 10

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Making TAE buffer

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Culture Innoculation

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Gemstone dye protocol

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Fractionation

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LB + Plate

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Supplemented Media

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Transformations

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Miniprep (Promega)

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OMV Purification

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PCR for Removal of His6 Tag from DsbA

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Sequencing

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2xYT

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Self-circularization of linear DNA

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Spectinmyocin

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Tyler's Transformation and Fluorescence

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