Team:Calgary/20 May 2009

From 2009.igem.org

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<div class="heading">NOTEBOOK PAGE INDEX</div>
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<div class="heading">THIS MONTH</div>
<div class="desc">
<div class="desc">
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<a href="#Carol">Carol</a><br>
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<a href="#Chinmoyee">Chinmoyee</a><br>
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</html>
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<a href="#Emily">Emily</a><br>
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{| style="background:transparent;"
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<a href="#Fahd">Fahd</a><br>
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|{{#calendar: query=preload=Team:Calgary/NotebookPreload | year=2009 | month=05 | title=Team:Calgary}}
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<a href="#Iman">Iman</a><br>
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|}
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<a href="#Jamie">Jamie</a><br>
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<a href="#Jeremy">Jeremy</a><br>
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<a href="#Katie">Katie</a><br>
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<a href="#Kevin">Kevin</a><br>
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<a href="#Mandy">Mandy</a><br>
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<a href="#Patrick">Patrick</a><br>
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<a href="#Prima">Prima</a><br>
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<a href="#Stefan">Stefan</a><br>
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<a href="#Vicki">Vicki</a><br>
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{{Template:CalgaryNotebook}}
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JULY 23, 2009 (DATE)
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MAY 20, 2009
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Amplification of <i>LUXCDABE</i> using Taq polymerase
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Amplification of <i>luxCDABE</i> using Taq polymerase
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The purpose of this experiment was to amplify ''LUXCDABE'' with Taq polymerase. Gene specific ''LUXCDABE'' forward and reverse primers were used. Conditions used were: Denature (94 Degrees Celsius, 3 minutes), 30 cycles (Denature (94 Degrees Celsius, 45 Seconds), Anneal (52 Degrees Celsius, 45 Seconds), Extend (72 Degrees Celsius, 6 minutes)), Final Extension (72 Degrees Celsius, 10 minutes), and hold at 4 Degrees Celsius overnight.  
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The purpose of this experiment was to amplify ''luxCDABE'' with Taq polymerase. Gene specific ''luxCDABE'' forward and reverse primers were used. Conditions used were: Denature (94 Degrees Celsius, 3 minutes), 30 cycles (Denature (94 Degrees Celsius, 45 Seconds), Anneal (52 Degrees Celsius, 45 Seconds), Extend (72 Degrees Celsius, 6 minutes)), Final Extension (72 Degrees Celsius, 10 minutes), and hold at 4 Degrees Celsius overnight.  
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Past Teams' Modelling
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WIKI CODING HERE
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Went through Other Team Wiki's to look for what they did with modelling
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Amplification of LuxOD47E
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WIKI CODING HERE
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Today we did a Polymerase Chain Reaction (PCR) with P Taq.  The purpose of this experiment was to amplify LuxOD47E . Gene specific LuxO forward and reverse primers were used. Conditions used were: Denature (94 Degrees Celsius, 3 minutes), 36 cycles (Denature (94 Degrees Celsius, 45 Seconds), Anneal (55 Degrees Celsius, 45 Seconds), Extend (72 Degrees Celsius, 2 minutes)), Final Extension (72 Degrees Celsius, 10 minutes), and hold at 4 Degrees Celsius overnight.
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FAHD
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WIKI CODING HERE
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IMAN
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JAMIE
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Amplification of LuxOD47A
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WIKI CODING HERE
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Purpose: Amplify LuxO D47A with pTaq PCR. LuxO-F/R primers were used fwith the following conditions: 94ºC for 3 minutes; 36 X (94ºC for 30 seconds; 55ºC for 45 seconds; 72ºC for 4 minutes); 72ºC for 10 minutes; held at 4ºC overnight.
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Planning the Second Life Project 
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WIKI CODING HERE
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Project Proposal was made and it appears the island will consist of three domains:
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* Biobrick Simulator
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* Synthetic Kingdom
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* Virtual Lab
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I have been assigned to the virtual lab and it will be mainly my responsibility to organize the activities that can be done in the building. A layout for the island is also in the process of being made.
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Making of different LB agar plates
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WIKI CODING HERE
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LB agar plates with different antibiotics were made for later use.
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Second Life Project Proposal
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WIKI CODING HERE
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We got together and made our project proposal, outlining the three areas of the island we wanted to develop, and a general idea of what would be in each. Katie and I are working on the Virtual Lab :). I walked through a couple more scripting tutorials and then did a lot of lab equipment building: shelves, tables, fridges, etc.
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<a name="Patrick"></a>
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<a name="Vicki"></a>
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PATRICK
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VICKI
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PCR Amplification of LuxPQ
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WIKI CODING HERE
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<b>Purpose:</b>
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To amplify LuxPQ on TOPO II Blunt on a PCR and verify that the sample really does consist of LuxPQ.
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<b>Materials and methods:</b>
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*DNA template: LuxPQ on TOPO II blunt
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PRIMA
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*Forward primer: LuxPQ forward (T_m = 60 degrees Celsius)
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*Reverse primer: LuxPQ reverse (T_m = 60 degrees Celsius)
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*Expected PCR product size: 3855 bp
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*DNA template concentration:
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**Colony 1: 254.7 ng/uL
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**Colony 9: 168.7 ng/uL
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2 amplification tubes were made for each colony, in addition to a negative control tube (for a total of 5 tubes)
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Master Mix contents (for 5 tubes):
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WIKI CODING HERE
 
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*10X PCR Buffer minus Mg 2+ (25 uL)
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*10mM dNTPs mixture (5 uL)
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*50mM MgCl2 (7.5 uL)
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*Forward primer [10 uM] (7.5 uL)
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*Reverse primer [10 uM] (7.5 uL)
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*Taq DNA polymerase [5 units/uL] (2.0 uL)
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*Autoclaved distilled HOH (190.5 uL)
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<b>TOTAL:</b> 245 uL of Master Mix, for 5 tubes of 49 uL each.
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Each PCR tube requires between 100-200 ng of template DNA. Accordingly, the Colony 1 sample was diluted to 200 ng/uL. 1 uL of DNA template was added to the following tubes:
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*Tube 1: Colony 1
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*Tube 2: Colony 1
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*Tube 3: Colony 9
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*Tube 4: Colony 9
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*Tube 5: dd HOH (negative control).
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PCR steps:
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*Denaturation: 94 degrees C, 3 minutes
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WIKI CODING HERE
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*Amplification: 36 cycles of:
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**Denaturation (94 degrees C, 45 seconds);
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**Annealing (55 degrees C, 45 seconds);
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**Extension (72 degrees C, 4 minutes)}
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*Final extension: 72 degrees C, 10 minutes
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*Hold temperature: 4 degrees C
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<b>Results:</b>
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None to offer here – the PCR tubes had mysteriously vanished from the PCR machine when I returned to collect them after the PCR had finished.
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Latest revision as of 07:35, 18 October 2009

University of Calgary

UNIVERSITY OF CALGARY



THIS MONTH

May
MTWTFSS
        [http://2009.igem.org/Team:Calgary/1_May_2009 1] [http://2009.igem.org/Team:Calgary/2_May_2009 2] [http://2009.igem.org/Team:Calgary/3_May_2009 3]
[http://2009.igem.org/Team:Calgary/4_May_2009 4] [http://2009.igem.org/Team:Calgary/5_May_2009 5] [http://2009.igem.org/Team:Calgary/6_May_2009 6] [http://2009.igem.org/Team:Calgary/7_May_2009 7] [http://2009.igem.org/Team:Calgary/8_May_2009 8] [http://2009.igem.org/Team:Calgary/9_May_2009 9] [http://2009.igem.org/Team:Calgary/10_May_2009 10]
[http://2009.igem.org/Team:Calgary/11_May_2009 11] [http://2009.igem.org/Team:Calgary/12_May_2009 12] [http://2009.igem.org/Team:Calgary/13_May_2009 13] [http://2009.igem.org/Team:Calgary/14_May_2009 14] [http://2009.igem.org/Team:Calgary/15_May_2009 15] [http://2009.igem.org/Team:Calgary/16_May_2009 16] [http://2009.igem.org/Team:Calgary/17_May_2009 17]
[http://2009.igem.org/Team:Calgary/18_May_2009 18] [http://2009.igem.org/Team:Calgary/19_May_2009 19] [http://2009.igem.org/Team:Calgary/20_May_2009 20] [http://2009.igem.org/Team:Calgary/21_May_2009 21] [http://2009.igem.org/Team:Calgary/22_May_2009 22] [http://2009.igem.org/Team:Calgary/23_May_2009 23] [http://2009.igem.org/Team:Calgary/24_May_2009 24]
[http://2009.igem.org/Team:Calgary/25_May_2009 25] [http://2009.igem.org/Team:Calgary/26_May_2009 26] [http://2009.igem.org/Team:Calgary/27_May_2009 27] [http://2009.igem.org/Team:Calgary/28_May_2009 28] [http://2009.igem.org/Team:Calgary/29_May_2009 29] [http://2009.igem.org/Team:Calgary/30_May_2009 30] [http://2009.igem.org/Team:Calgary/31_May_2009 31]


NOTEBOOK PAGE INDEX
Carol
Chinmoyee
Emily
Fahd
Iman
Jamie
Jeremy
Katie
Kevin
Mandy
Patrick
Prima
Stefan
Vicki



CALENDAR

May
MTWTFSS
        [http://2009.igem.org/Team:Calgary/1_May_2009 1] [http://2009.igem.org/Team:Calgary/2_May_2009 2] [http://2009.igem.org/Team:Calgary/3_May_2009 3]
[http://2009.igem.org/Team:Calgary/4_May_2009 4] [http://2009.igem.org/Team:Calgary/5_May_2009 5] [http://2009.igem.org/Team:Calgary/6_May_2009 6] [http://2009.igem.org/Team:Calgary/7_May_2009 7] [http://2009.igem.org/Team:Calgary/8_May_2009 8] [http://2009.igem.org/Team:Calgary/9_May_2009 9] [http://2009.igem.org/Team:Calgary/10_May_2009 10]
[http://2009.igem.org/Team:Calgary/11_May_2009 11] [http://2009.igem.org/Team:Calgary/12_May_2009 12] [http://2009.igem.org/Team:Calgary/13_May_2009 13] [http://2009.igem.org/Team:Calgary/14_May_2009 14] [http://2009.igem.org/Team:Calgary/15_May_2009 15] [http://2009.igem.org/Team:Calgary/16_May_2009 16] [http://2009.igem.org/Team:Calgary/17_May_2009 17]
[http://2009.igem.org/Team:Calgary/18_May_2009 18] [http://2009.igem.org/Team:Calgary/19_May_2009 19] [http://2009.igem.org/Team:Calgary/20_May_2009 20] [http://2009.igem.org/Team:Calgary/21_May_2009 21] [http://2009.igem.org/Team:Calgary/22_May_2009 22] [http://2009.igem.org/Team:Calgary/23_May_2009 23] [http://2009.igem.org/Team:Calgary/24_May_2009 24]
[http://2009.igem.org/Team:Calgary/25_May_2009 25] [http://2009.igem.org/Team:Calgary/26_May_2009 26] [http://2009.igem.org/Team:Calgary/27_May_2009 27] [http://2009.igem.org/Team:Calgary/28_May_2009 28] [http://2009.igem.org/Team:Calgary/29_May_2009 29] [http://2009.igem.org/Team:Calgary/30_May_2009 30] [http://2009.igem.org/Team:Calgary/31_May_2009 31]


June
MTWTFSS
[http://2009.igem.org/Team:Calgary/1_June_2009 1] [http://2009.igem.org/Team:Calgary/2_June_2009 2] [http://2009.igem.org/Team:Calgary/3_June_2009 3] [http://2009.igem.org/Team:Calgary/4_June_2009 4] [http://2009.igem.org/Team:Calgary/5_June_2009 5] [http://2009.igem.org/Team:Calgary/6_June_2009 6] [http://2009.igem.org/Team:Calgary/7_June_2009 7]
[http://2009.igem.org/Team:Calgary/8_June_2009 8] [http://2009.igem.org/Team:Calgary/9_June_2009 9] [http://2009.igem.org/Team:Calgary/10_June_2009 10] [http://2009.igem.org/Team:Calgary/11_June_2009 11] [http://2009.igem.org/Team:Calgary/12_June_2009 12] [http://2009.igem.org/Team:Calgary/13_June_2009 13] [http://2009.igem.org/Team:Calgary/14_June_2009 14]
[http://2009.igem.org/Team:Calgary/15_June_2009 15] [http://2009.igem.org/Team:Calgary/16_June_2009 16] [http://2009.igem.org/Team:Calgary/17_June_2009 17] [http://2009.igem.org/Team:Calgary/18_June_2009 18] [http://2009.igem.org/Team:Calgary/19_June_2009 19] [http://2009.igem.org/Team:Calgary/20_June_2009 20] [http://2009.igem.org/Team:Calgary/21_June_2009 21]
[http://2009.igem.org/Team:Calgary/22_June_2009 22] [http://2009.igem.org/Team:Calgary/23_June_2009 23] [http://2009.igem.org/Team:Calgary/24_June_2009 24] [http://2009.igem.org/Team:Calgary/25_June_2009 25] [http://2009.igem.org/Team:Calgary/26_June_2009 26] [http://2009.igem.org/Team:Calgary/27_June_2009 27] [http://2009.igem.org/Team:Calgary/28_June_2009 28]
[http://2009.igem.org/Team:Calgary/29_June_2009 29] [http://2009.igem.org/Team:Calgary/30_June_2009 30]


July
MTWTFSS
    [http://2009.igem.org/Team:Calgary/1_July_2009 1] [http://2009.igem.org/Team:Calgary/2_July_2009 2] [http://2009.igem.org/Team:Calgary/3_July_2009 3] [http://2009.igem.org/Team:Calgary/4_July_2009 4] [http://2009.igem.org/Team:Calgary/5_July_2009 5]
[http://2009.igem.org/Team:Calgary/6_July_2009 6] [http://2009.igem.org/Team:Calgary/7_July_2009 7] [http://2009.igem.org/Team:Calgary/8_July_2009 8] [http://2009.igem.org/Team:Calgary/9_July_2009 9] [http://2009.igem.org/Team:Calgary/10_July_2009 10] [http://2009.igem.org/Team:Calgary/11_July_2009 11] [http://2009.igem.org/Team:Calgary/12_July_2009 12]
[http://2009.igem.org/Team:Calgary/13_July_2009 13] [http://2009.igem.org/Team:Calgary/14_July_2009 14] [http://2009.igem.org/Team:Calgary/15_July_2009 15] [http://2009.igem.org/Team:Calgary/16_July_2009 16] [http://2009.igem.org/Team:Calgary/17_July_2009 17] [http://2009.igem.org/Team:Calgary/18_July_2009 18] [http://2009.igem.org/Team:Calgary/19_July_2009 19]
[http://2009.igem.org/Team:Calgary/20_July_2009 20] [http://2009.igem.org/Team:Calgary/21_July_2009 21] [http://2009.igem.org/Team:Calgary/22_July_2009 22] [http://2009.igem.org/Team:Calgary/23_July_2009 23] [http://2009.igem.org/Team:Calgary/24_July_2009 24] [http://2009.igem.org/Team:Calgary/25_July_2009 25] [http://2009.igem.org/Team:Calgary/26_July_2009 26]
[http://2009.igem.org/Team:Calgary/27_July_2009 27] [http://2009.igem.org/Team:Calgary/28_July_2009 28] [http://2009.igem.org/Team:Calgary/29_July_2009 29] [http://2009.igem.org/Team:Calgary/30_July_2009 30] [http://2009.igem.org/Team:Calgary/31_July_2009 31]


August
MTWTFSS
          [http://2009.igem.org/Team:Calgary/1_August_2009 1] [http://2009.igem.org/Team:Calgary/2_August_2009 2]
[http://2009.igem.org/Team:Calgary/3_August_2009 3] [http://2009.igem.org/Team:Calgary/4_August_2009 4] [http://2009.igem.org/Team:Calgary/5_August_2009 5] [http://2009.igem.org/Team:Calgary/6_August_2009 6] [http://2009.igem.org/Team:Calgary/7_August_2009 7] [http://2009.igem.org/Team:Calgary/8_August_2009 8] [http://2009.igem.org/Team:Calgary/9_August_2009 9]
[http://2009.igem.org/Team:Calgary/10_August_2009 10] [http://2009.igem.org/Team:Calgary/11_August_2009 11] [http://2009.igem.org/Team:Calgary/12_August_2009 12] [http://2009.igem.org/Team:Calgary/13_August_2009 13] [http://2009.igem.org/Team:Calgary/14_August_2009 14] [http://2009.igem.org/Team:Calgary/15_August_2009 15] [http://2009.igem.org/Team:Calgary/16_August_2009 16]
[http://2009.igem.org/Team:Calgary/17_August_2009 17] [http://2009.igem.org/Team:Calgary/18_August_2009 18] [http://2009.igem.org/Team:Calgary/19_August_2009 19] [http://2009.igem.org/Team:Calgary/20_August_2009 20] [http://2009.igem.org/Team:Calgary/21_August_2009 21] [http://2009.igem.org/Team:Calgary/22_August_2009 22] [http://2009.igem.org/Team:Calgary/23_August_2009 23]
[http://2009.igem.org/Team:Calgary/24_August_2009 24] [http://2009.igem.org/Team:Calgary/25_August_2009 25] [http://2009.igem.org/Team:Calgary/26_August_2009 26] [http://2009.igem.org/Team:Calgary/27_August_2009 27] [http://2009.igem.org/Team:Calgary/28_August_2009 28] [http://2009.igem.org/Team:Calgary/29_August_2009 29] [http://2009.igem.org/Team:Calgary/30_August_2009 30]
[http://2009.igem.org/Team:Calgary/31_August_2009 31]


September
MTWTFSS
  [http://2009.igem.org/Team:Calgary/1_September_2009 1] [http://2009.igem.org/Team:Calgary/2_September_2009 2] [http://2009.igem.org/Team:Calgary/3_September_2009 3] [http://2009.igem.org/Team:Calgary/4_September_2009 4] [http://2009.igem.org/Team:Calgary/5_September_2009 5] [http://2009.igem.org/Team:Calgary/6_September_2009 6]
[http://2009.igem.org/Team:Calgary/7_September_2009 7] [http://2009.igem.org/Team:Calgary/8_September_2009 8] [http://2009.igem.org/Team:Calgary/9_September_2009 9] [http://2009.igem.org/Team:Calgary/10_September_2009 10] [http://2009.igem.org/Team:Calgary/11_September_2009 11] [http://2009.igem.org/Team:Calgary/12_September_2009 12] [http://2009.igem.org/Team:Calgary/13_September_2009 13]
[http://2009.igem.org/Team:Calgary/14_September_2009 14] [http://2009.igem.org/Team:Calgary/15_September_2009 15] [http://2009.igem.org/Team:Calgary/16_September_2009 16] [http://2009.igem.org/Team:Calgary/17_September_2009 17] [http://2009.igem.org/Team:Calgary/18_September_2009 18] [http://2009.igem.org/Team:Calgary/19_September_2009 19] [http://2009.igem.org/Team:Calgary/20_September_2009 20]
[http://2009.igem.org/Team:Calgary/21_September_2009 21] [http://2009.igem.org/Team:Calgary/22_September_2009 22] [http://2009.igem.org/Team:Calgary/23_September_2009 23] [http://2009.igem.org/Team:Calgary/24_September_2009 24] [http://2009.igem.org/Team:Calgary/25_September_2009 25] [http://2009.igem.org/Team:Calgary/26_September_2009 26] [http://2009.igem.org/Team:Calgary/27_September_2009 27]
[http://2009.igem.org/Team:Calgary/28_September_2009 28] [http://2009.igem.org/Team:Calgary/29_September_2009 29] [http://2009.igem.org/Team:Calgary/30_September_2009 30]


October
MTWTFSS
      [http://2009.igem.org/Team:Calgary/1_October_2009 1] [http://2009.igem.org/Team:Calgary/2_October_2009 2] [http://2009.igem.org/Team:Calgary/3_October_2009 3] [http://2009.igem.org/Team:Calgary/4_October_2009 4]
[http://2009.igem.org/Team:Calgary/5_October_2009 5] [http://2009.igem.org/Team:Calgary/6_October_2009 6] [http://2009.igem.org/Team:Calgary/7_October_2009 7] [http://2009.igem.org/Team:Calgary/8_October_2009 8] [http://2009.igem.org/Team:Calgary/9_October_2009 9] [http://2009.igem.org/Team:Calgary/10_October_2009 10] [http://2009.igem.org/Team:Calgary/11_October_2009 11]
[http://2009.igem.org/Team:Calgary/12_October_2009 12] [http://2009.igem.org/Team:Calgary/13_October_2009 13] [http://2009.igem.org/Team:Calgary/14_October_2009 14] [http://2009.igem.org/Team:Calgary/15_October_2009 15] [http://2009.igem.org/Team:Calgary/16_October_2009 16] [http://2009.igem.org/Team:Calgary/17_October_2009 17] [http://2009.igem.org/Team:Calgary/18_October_2009 18]
[http://2009.igem.org/wiki/index.php?title=Team:Calgary/19_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/20_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/21_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/22_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/23_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/24_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/25_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 25]
[http://2009.igem.org/wiki/index.php?title=Team:Calgary/26_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/27_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/28_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/29_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/30_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/31_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 31]



MAY 20, 2009


CAROL

Amplification of luxCDABE using Taq polymerase

The purpose of this experiment was to amplify luxCDABE with Taq polymerase. Gene specific luxCDABE forward and reverse primers were used. Conditions used were: Denature (94 Degrees Celsius, 3 minutes), 30 cycles (Denature (94 Degrees Celsius, 45 Seconds), Anneal (52 Degrees Celsius, 45 Seconds), Extend (72 Degrees Celsius, 6 minutes)), Final Extension (72 Degrees Celsius, 10 minutes), and hold at 4 Degrees Celsius overnight.


CHINMOYEE

Past Teams' Modelling

Went through Other Team Wiki's to look for what they did with modelling


EMILY

Amplification of LuxOD47E

Today we did a Polymerase Chain Reaction (PCR) with P Taq. The purpose of this experiment was to amplify LuxOD47E . Gene specific LuxO forward and reverse primers were used. Conditions used were: Denature (94 Degrees Celsius, 3 minutes), 36 cycles (Denature (94 Degrees Celsius, 45 Seconds), Anneal (55 Degrees Celsius, 45 Seconds), Extend (72 Degrees Celsius, 2 minutes)), Final Extension (72 Degrees Celsius, 10 minutes), and hold at 4 Degrees Celsius overnight.


JEREMY

Amplification of LuxOD47A

Purpose: Amplify LuxO D47A with pTaq PCR. LuxO-F/R primers were used fwith the following conditions: 94ºC for 3 minutes; 36 X (94ºC for 30 seconds; 55ºC for 45 seconds; 72ºC for 4 minutes); 72ºC for 10 minutes; held at 4ºC overnight.


KATIE

Planning the Second Life Project

Project Proposal was made and it appears the island will consist of three domains:

  • Biobrick Simulator
  • Synthetic Kingdom
  • Virtual Lab

I have been assigned to the virtual lab and it will be mainly my responsibility to organize the activities that can be done in the building. A layout for the island is also in the process of being made.


KEVIN

Making of different LB agar plates

LB agar plates with different antibiotics were made for later use.


MANDY

Second Life Project Proposal

We got together and made our project proposal, outlining the three areas of the island we wanted to develop, and a general idea of what would be in each. Katie and I are working on the Virtual Lab :). I walked through a couple more scripting tutorials and then did a lot of lab equipment building: shelves, tables, fridges, etc.

VICKI

PCR Amplification of LuxPQ

Purpose:
To amplify LuxPQ on TOPO II Blunt on a PCR and verify that the sample really does consist of LuxPQ.

Materials and methods:

  • DNA template: LuxPQ on TOPO II blunt
  • Forward primer: LuxPQ forward (T_m = 60 degrees Celsius)
  • Reverse primer: LuxPQ reverse (T_m = 60 degrees Celsius)
  • Expected PCR product size: 3855 bp
  • DNA template concentration:
    • Colony 1: 254.7 ng/uL
    • Colony 9: 168.7 ng/uL


2 amplification tubes were made for each colony, in addition to a negative control tube (for a total of 5 tubes)
Master Mix contents (for 5 tubes):

  • 10X PCR Buffer minus Mg 2+ (25 uL)
  • 10mM dNTPs mixture (5 uL)
  • 50mM MgCl2 (7.5 uL)
  • Forward primer [10 uM] (7.5 uL)
  • Reverse primer [10 uM] (7.5 uL)
  • Taq DNA polymerase [5 units/uL] (2.0 uL)
  • Autoclaved distilled HOH (190.5 uL)


TOTAL: 245 uL of Master Mix, for 5 tubes of 49 uL each.

Each PCR tube requires between 100-200 ng of template DNA. Accordingly, the Colony 1 sample was diluted to 200 ng/uL. 1 uL of DNA template was added to the following tubes:

  • Tube 1: Colony 1
  • Tube 2: Colony 1
  • Tube 3: Colony 9
  • Tube 4: Colony 9
  • Tube 5: dd HOH (negative control).


PCR steps:

  • Denaturation: 94 degrees C, 3 minutes
  • Amplification: 36 cycles of:
    • Denaturation (94 degrees C, 45 seconds);
    • Annealing (55 degrees C, 45 seconds);
    • Extension (72 degrees C, 4 minutes)}
  • Final extension: 72 degrees C, 10 minutes
  • Hold temperature: 4 degrees C

Results: None to offer here – the PCR tubes had mysteriously vanished from the PCR machine when I returned to collect them after the PCR had finished.

Retrieved from "http://2009.igem.org/Team:Calgary/20_May_2009"