Team:Calgary/22 June 2009

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June 22, 2009
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JUNE 22, 2009
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Colony PCR and Enzyme Digestion of Mutated Plasmid
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WIKI CODING HERE
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* Attempted to perform colony PCR and enzyme digestion with XbaI on the mutated plasmid with a positive control (non-mutated plasmid)
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* Results showed that plasmid does NOT contain the XbaI site (the 0.6% agarose gel showed that there is only one 6KB band from the colonies whereas the positive control showed two bands)
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* Will try to isolate higher concentrations for sequencing.
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Research more on Stochasticity
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Looked into Papers :
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*Imapct of Stochasticity on gene regulatory networks
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*Analysis of Noise in AI-2 Circuits
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*Stochasticity in Transcriptional Regulation: Origins, Consequences,and Mathematical Representations
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*Purpose: To verify that BBK LuxOD47E has been successfully cloned into the psB1AC3 vector.
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*Performed a Colony PCR on LuxOD47E in psB1AC3 vector colonies from last week.  Only digests done with XbaI / PstI enzymes produced colonies on plates, so these were the only colonies used.
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*Used p Taq and LuxO forward and reverse primers.  Cycling conditions were as follows: 94 C for 6 minutes, 36x (94 C for 30 sec, 55 C for 45 sec, 72 C for 90 sec), 72 C for 10 minutes and hold at 4 C.
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*See gel photo below.  Lanes 1-6 are LuxOD47E cut with XbaI / PstI, colonies 1-6, Lane 7 is a negative control with ddH2O.
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[[Image:2009.06.22ColonyPCR1.jpg|350px]]
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*Results:  Nothing was amplified.  This was because I used the wrong buffer.  Will proceed by re-running the Colony PCR with the CORRECT buffer (10x PCR Buffer- Cl2).
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*Performed colony PCR again with the proper PCR Buffer this time. PCR products were visualized on a 1% agarose gel run at 120 V with Generuler 1.0 KB+ DNA Ladder. Lanes 1-7 are LuxOD47E in psB1AC3 cut with XbaI / PstI. Lane 8 is a negative control with only Mastermix + ddH2O.
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*See gel photo below.
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[[Image:2009.06.23ColonyPCR2.jpg|350px]]
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*From this gel, it looks like LuxOD47E is in the vector as we see the appropriate band size of approximately 1.4 KB.
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*Prepared overnight cultures of the colonies, will isolate plasmid tomorrow.
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IMAN
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Construction verification
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WIKI CODING HERE
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Colony PCR could not be used as a means to verify due to the presence of intrinsic terminators (B0015) within the plasmid backbone.  If B0015 gene-specific primers are used, multiple bands will likely appear.  Instead a ''Not''I digest coupled with a size control will be used as a verification. Standard manufacturer's specifications followed.
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PCR of luxPQ in psB1AK3 using BBK-CP-F/R and gene specific primers
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Purpose: to verify presence of LuxPQ in psB1AK3. Colony 4 of luxPQ in psB1AK3 was used as a template and pTaq DNA polymerase was used. LuxPQ-F/R gene specific primers and BBK-CP-F/R primers (that anneal just outside the BBK prefix and suffix) were used. Conditions were as follows: 94ºC for 2 minutes; 36X (94ºC for 30 seconds; 55ºC for 45 seconds for BBK-CP-F/R primers (53ºC for 45 seconds for LuxPQ F/R primers); 72ºC for 4 minutes); 72ºC for 10 minutes; held at 4ºC. The products were then run on a 0.8% agarose gel (see below). Again, we are looking for a band size of just under 4kb, which we do not see here, and we must thus restart from luxPQ in TOPO.
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[[Image:2009.06.22.LuxPQ_BBKCP_BBK.png|700px]]
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Restreak of R0040, B0015, and J13002 were done to grow more of verified single colonies.
Restreak of R0040, B0015, and J13002 were done to grow more of verified single colonies.
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[[Image:Calgary_2009.06.16.B0015_R0040_J13002.plates.png‎]]
 
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Understanding Lab and Continuation with marketing
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WIKI CODING HERE
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I learned the purpose and procedures for restriction digest from shadowing Jeremy. After hanging out with in the lab for some time, I began understanding what PCR was used for, when PCR needed to be done, what's the water bath for and much more.
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Carol also taught me what phosphatase is, when it should be done and even showed me how she was doing it.
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I kept following-up with the list of companies that Fahd and I had made in the beginning of the year. Many companies have turned down our proposals due to financial constraints. On the bright side, we decided to target mostly pharmaceutical and biotech companies, many of which are considering our sponsorship proposal.
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Latest revision as of 06:25, 20 October 2009

University of Calgary

UNIVERSITY OF CALGARY



THIS MONTH

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]


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]



JUNE 22, 2009


CAROL

Colony PCR and Enzyme Digestion of Mutated Plasmid

  • Attempted to perform colony PCR and enzyme digestion with XbaI on the mutated plasmid with a positive control (non-mutated plasmid)
  • Results showed that plasmid does NOT contain the XbaI site (the 0.6% agarose gel showed that there is only one 6KB band from the colonies whereas the positive control showed two bands)
  • Will try to isolate higher concentrations for sequencing.


CHINMOYEE

Research more on Stochasticity

Looked into Papers :

  • Imapct of Stochasticity on gene regulatory networks
  • Analysis of Noise in AI-2 Circuits
  • Stochasticity in Transcriptional Regulation: Origins, Consequences,and Mathematical Representations


EMILY

Descriptive Title of What You're Doing

  • Purpose: To verify that BBK LuxOD47E has been successfully cloned into the psB1AC3 vector.
  • Performed a Colony PCR on LuxOD47E in psB1AC3 vector colonies from last week. Only digests done with XbaI / PstI enzymes produced colonies on plates, so these were the only colonies used.
  • Used p Taq and LuxO forward and reverse primers. Cycling conditions were as follows: 94 C for 6 minutes, 36x (94 C for 30 sec, 55 C for 45 sec, 72 C for 90 sec), 72 C for 10 minutes and hold at 4 C.
  • See gel photo below. Lanes 1-6 are LuxOD47E cut with XbaI / PstI, colonies 1-6, Lane 7 is a negative control with ddH2O.


2009.06.22ColonyPCR1.jpg

  • Results: Nothing was amplified. This was because I used the wrong buffer. Will proceed by re-running the Colony PCR with the CORRECT buffer (10x PCR Buffer- Cl2).
  • Performed colony PCR again with the proper PCR Buffer this time. PCR products were visualized on a 1% agarose gel run at 120 V with Generuler 1.0 KB+ DNA Ladder. Lanes 1-7 are LuxOD47E in psB1AC3 cut with XbaI / PstI. Lane 8 is a negative control with only Mastermix + ddH2O.
  • See gel photo below.


2009.06.23ColonyPCR2.jpg

  • From this gel, it looks like LuxOD47E is in the vector as we see the appropriate band size of approximately 1.4 KB.
  • Prepared overnight cultures of the colonies, will isolate plasmid tomorrow.


JAMIE

Construction verification

Colony PCR could not be used as a means to verify due to the presence of intrinsic terminators (B0015) within the plasmid backbone. If B0015 gene-specific primers are used, multiple bands will likely appear. Instead a NotI digest coupled with a size control will be used as a verification. Standard manufacturer's specifications followed.


JEREMY

PCR of luxPQ in psB1AK3 using BBK-CP-F/R and gene specific primers

Purpose: to verify presence of LuxPQ in psB1AK3. Colony 4 of luxPQ in psB1AK3 was used as a template and pTaq DNA polymerase was used. LuxPQ-F/R gene specific primers and BBK-CP-F/R primers (that anneal just outside the BBK prefix and suffix) were used. Conditions were as follows: 94ºC for 2 minutes; 36X (94ºC for 30 seconds; 55ºC for 45 seconds for BBK-CP-F/R primers (53ºC for 45 seconds for LuxPQ F/R primers); 72ºC for 4 minutes); 72ºC for 10 minutes; held at 4ºC. The products were then run on a 0.8% agarose gel (see below). Again, we are looking for a band size of just under 4kb, which we do not see here, and we must thus restart from luxPQ in TOPO.
2009.06.22.LuxPQ BBKCP BBK.png


KEVIN

Restreak

Restreak of R0040, B0015, and J13002 were done to grow more of verified single colonies.



PRIMA

Understanding Lab and Continuation with marketing

I learned the purpose and procedures for restriction digest from shadowing Jeremy. After hanging out with in the lab for some time, I began understanding what PCR was used for, when PCR needed to be done, what's the water bath for and much more.

Carol also taught me what phosphatase is, when it should be done and even showed me how she was doing it. I kept following-up with the list of companies that Fahd and I had made in the beginning of the year. Many companies have turned down our proposals due to financial constraints. On the bright side, we decided to target mostly pharmaceutical and biotech companies, many of which are considering our sponsorship proposal.

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