Team:Queens Canada/Notebook/Week10

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<p>Notebook - Week 9</p>
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<p>Notebook - Week 10</p>
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Week
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<li> <a href="http://2012.igem.org/Team:Queens_Canada/Notebook/Week1">1</a> </li>
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<li> <a href="http://2012.igem.org/Team:Queens_Canada/Notebook/Week2">2</a> </li>
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<li> <a href="http://2012.igem.org/Team:Queens_Canada/Notebook/Week3">3</a> </li>
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<li> <a href="http://2012.igem.org/Team:Queens_Canada/Notebook/Week4">4</a> </li>
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<li> <a href="http://2012.igem.org/Team:Queens_Canada/Notebook/Week5">5</a> </li>
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<li> <a href="http://2012.igem.org/Team:Queens_Canada/Notebook/Week6">6</a> </li>
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<li> <a href="http://2012.igem.org/Team:Queens_Canada/Notebook/Week7">7</a> </li>
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<li> <a href="http://2012.igem.org/Team:Queens_Canada/Notebook/Week8">8</a> </li>
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<li> <a href="http://2012.igem.org/Team:Queens_Canada/Notebook/Week9">9</a> </li>
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<li> <a href="http://2012.igem.org/Team:Queens_Canada/Notebook/Week10">10</a> </li>
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<li> <a href="http://2012.igem.org/Team:Queens_Canada/Notebook/Week11">11</a> </li>
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<li> <a href="http://2012.igem.org/Team:Queens_Canada/Notebook/Week12">12</a> </li>
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<li> <a href="http://2012.igem.org/Team:Queens_Canada/Notebook/Week13">13</a> </li>
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<li> <a href="http://2012.igem.org/Team:Queens_Canada/Notebook/Week14">14</a> </li>
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<li> <a href="http://2012.igem.org/Team:Queens_Canada/Notebook/Week15">15</a> </li>
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<li> <a href="http://2012.igem.org/Team:Queens_Canada/Notebook/Week16">16</a> </li>
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<li> <a href="http://2012.igem.org/Team:Queens_Canada/Notebook/Week17">17+</a> </li>
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        <div id="protocolcontent"><table class="tableizer-table">
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<tr class="tableizer-firstrow"><th>Date</th><th>Protocol</th><th>People</th><th>DNA (if relevant)</th><th>Quantities and Parameters (if relevant)</th><th>Notes on Protocol/ Gel Lanes</th></tr> <tr><td>Jul-03</td><td>Digestion and Ligation</td><td>Dan, Victor, Beini</td><td>pSB1C3 and GFP + fliC2 pSB1C3</td><td>Digestion Mix: (GFP/fliC2)<br>Eco RI - HF / XbaI<br>SpeI / PstI<br>10X NEB Buffer 2<br>100X BSA<br>H20 </td><td>&nbsp;</td></tr> <tr><td>&nbsp;</td><td>Streak Plates of competent cells</td><td>&nbsp;</td><td>BL21 DE3 plyss [C] 2 trials<br>XLI Blue [tet] 2 trials<br>DH5 - alpha [nal] 2 trials</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>&nbsp;</td><td>Heat shock transformation</td><td>Dan,</td><td>FliC C2 in Kanamycin </td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>&nbsp;</td><td>Ligation </td><td>David</td><td>fmT PCR product into plasmid 1A3</td><td>Ligation Mix:<br>2 ul PCR Product<br>1 ul EcoRI<br>1 ul PstI<br>5 ul NEBuffer 2<br>0.5 BSA <br>40.5 Water<br><br>Plasmid: 4 ul plasmid + 4 ul mastermix </td><td>&nbsp;</td></tr> <tr><td>&nbsp;</td><td>Heat shock transformation</td><td>Andrew</td><td>5 parts:<br>GFP + fliC C2 + psB1C3 (two trials)<br>GFP + pSB1C3<br>fliC2 + pSB1C3<br>fmt + pSB1A3</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>Jul-04</td><td>Digestion and Ligation</td><td>&nbsp;</td><td>1)GFP, 2) GFP, 3) GFP, 1) fmT, 2) fmT, 3) fmT</td><td>Digestion Protocol (NEB):<br>NEB2: 2.5 ul<br>BSA: 0.5 ul<br>EcoRI: 0.5 ul<br>PstI : 0.5 ul<br><br>Used parts registry protocol for digestion:<br>1)GFP: DNA 10 ul, H20 6 ul, 2) GFP: DNA 5 ul, H20: 11 ul 3)GFP: DNA 2 ul, H20 14 ul, 1) fmT: DNA 10 ul, H20: 6 ul, 2)fmt: DNA 5 ul, H20 11 ul, 3) fmT: DNA 2 ul, H20 14 ul</td><td>&nbsp;</td></tr> <tr><td>&nbsp;</td><td>Liquid Cultures</td><td>&nbsp;</td><td>XLI Blue, BL21 DE5 Plys, DH5&#945;, 2x fliC kanamycin,  </td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>&nbsp;</td><td>Ligations</td><td>&nbsp;</td><td>6 Ligations:<br>1) GFP + pSB1C3 ligation T1<br>2) GFP + pSB1C3 ligation T2<br>3) GFP + pSB1C3 ligation T3<br>4) fmt + pSB1A3 ligation T1<br>5) fmt + pSB1A3 ligation T2<br>6) fmt + pSB1A3 ligation T3</td><td>&nbsp;</td><td>Protocol was not recorded, NEB protocol was used.</td></tr> <tr><td>Jul-05</td><td>Miniprep</td><td>Victor, Kevin, Beini</td><td>fliC C2 x3 </td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>&nbsp;</td><td>Genomic DNA Isolation</td><td>&nbsp;</td><td>XLI Blue, BL21 DE5 Plys, DH5&#945;.</td><td>&nbsp;</td><td>70% Ethanol required for the protocol <br>was diluted using 85% Ethanol.</td></tr> <tr><td>&nbsp;</td><td>Primer Resuspension and <br>dilutions</td><td>David</td><td>Metal-Binding Part primers:<br>Primer resuspension and dilutions for GoIB, SmtA and MBP<br></td><td>&nbsp;</td><td>note: MBP LP may not have exact amount of TE added during resuspension due to pipetting error.<br>Note: SmTA LP dilution may not have enough primer added --> If PCR failts try making another dilution</td></tr> <tr><td>Jul-06</td><td>PCR</td><td>David, Andrew</td><td>PbrR, SmtA, GolB<br>Four trials of each + one control each<br></td><td>Mastermix<br>Thermopol Rxn Buffer: 5 ul<br>DNTPs (10 mM): 1 ul<br>LP: 1.5 ul<br>RP: 1.5 ul<br>DNA Template: 5 ul<br>Vent Polymerase: 0.5 ul<br>MgSO4: 1 ul<br>H2O: 34.5 ul<br>Total: 50 ul<br>Program:1. Denaturation (Initial): 95  for 5 mins 2. Denaturation: 95C for 20 seconds 3. Annealing: 60C for 15 seconds  4. Extension: 72 degrees, 30 seconds  5. Repeat step 2-4 for 30 cycles  6. Final extension 72 degrees, 5 minute  7. Cool down 4 degrees, one hour</td><td>Big Gel:<br>1) Ladder, 2)mCit FD digest EP, 3) PbrR Control<br>4) PbrR Trial 1, 5) PbrR Trial 2, 6) PbrR Trial 3, 7) PbrR Trial 4, 8) Ladder, 9) SmtA Control, 10) SmtA Trial 1, 11) SmtA Trial 2, 12) SmtA Trial 3, 13) SmtA Trial 4, 14) SmtA Trial 5, 15) Ladder, 16) GoIB Control, 17) GoIB Trial 1, 18) GoIB Trial 2, 19) GoIB Trial 3, 20) GoIB Trial 4, 21) GoIB Trial 5, 22) Ladder, 23) PbrR Trial 5.</td></tr> <tr><td>&nbsp;</td><td>NEB Protocol Digestion</td><td>Victor</td><td>XLI Blue, BL21 DE5 Plys, DH5&#945;, fliC C2, Luciferase, mCitrine, </td><td>&nbsp;</td><td>Small Gel:<br>1) Ladder, 2) DH5&#945; NEB EP Digest,3)  DH5&#945; Control, 4) XLI Control, 5) LI Digest Blue NEB EP, 6) BL21 Control, 7) BL21 Fast Digest EP, 8) fliC C3 Digest NEB EP, 9) Ladder,10) fliC C2 Control, 11) fliC C2 digest FD EP, 12) Luciferase Control, 13) Luciferase NEB T1 EP, 14) Luciferase NEB T2 EP, 15) mCitrine Control, 16) mCit FD Digest EP.</td></tr> <tr><td>&nbsp;</td><td>Heat Shock Transformation</td><td>Andrew</td><td>Parts list BBa_:<br>J04500 - Promoter, J13002 - Promoter, 1712074 - Promoter, B0010 - Terminator, B0015 - Terminator, B0017 - Terminator, B0030 - Strong RBS, B0031 - Weak RBS, B0032 - Medium RBS</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>Jul-06</td><td>PCR (Thermopol Protocol)</td><td>Kevin</td><td>Four trials of each strain + one control</td><td>Thermopol Protocol:<br>Thermopol: 5 ul<br>dNTPs: 1 ul<br>LP: 1.5 ul<br>RP: 1.5 ul<br>DNA Template: 5 ul<br>Vent Pol: 0.5 ul<br>MgSO4: 1 ul<br>H2O: 34.5 ul<br><br>Program:1. Denaturation (Initial): 95  for 5 mins 2. Denaturation: 98C for 20 seconds 3. Annealing: 55C for 15 seconds  4. Extension: 72 degrees, 30 seconds  5. Repeat step 2-4 for 30 cycles  6. Final extension 72 degrees, 5 minute  7. Cool down 4 degrees, one hour</td><td>&nbsp;</td></tr> <tr><td>Jul-07</td><td>Miniprep</td><td>Kevin, Maggie</td><td>fmT+ pSB1C3, GFP + pSB1C3 (2 trials each)</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>&nbsp;</td><td>Digestions</td><td>&nbsp;</td><td>Using Trial #1 from above minipreps</td><td>NEB Digestion Protocol:<br> 2 ul Templates<br>1 ul EcoRI HF <br>1 ul SpeI (NEB)<br>5 ul NEB Buffer 2<br>0.5 BSA <br>40.5 H20<br><br>Fast Digest Protocol:<br>2 ul Template<br>2 ul 10x FD Buffer Green<br>1 ul SpeI (FD)<br>1 ul EcoRI (NEB)<br>14 ul H20</td><td>&nbsp;</td></tr> <tr><td>&nbsp;</td><td>Gel Electrophoresis</td><td>&nbsp;</td><td>28 Lanes total</td><td>&nbsp;</td><td>Large Gel:<br>1) Ladder, 2) Control (fliC), 3) XLI Blue T1<br>4) XLI Blue T2, 5) XLI Blue T3, 6) XLI Blue T4<br>7) Ladder, 8) DH5&#945; T1, 9) DH5&#945; T2, 10) DH5&#945; T3, 11) DH5&#945; T4, 12) Blank, 13) Blank, 14) Blank, 15) Ladder, 16) BL 21 T1, 17) BL 21 T2, 18) BL 21 T3, 19) BL 21 T4, 20) fmT mpp T1, 21) Ladder, 22) fmT NEB, 23) fmT FD (Green) 24) GFP Mpp, 28) GFP NEB, 29) GFP FD (Green)</td></tr> <tr><td>Jul-08</td><td>PCR</td><td>Kevin, Maggie</td><td>XL1 Blue</td><td>PCR using XLI Blue:<br>Buffer 5 ul<br>DNTPs 1 ul<br>LP 1.5 ul<br>Rp 1.5 ul<br>Template 5 ul<br>Vent 0.5 ul<br>MgSO4 1 ul<br>H20 34.5 ul<br>Program:1. Denaturation (Initial): 95  for 5 mins 2. Denaturation: 98C for 20 seconds 3. Annealing: 55C for 15 seconds  4. Extension: 72 degrees, 30 seconds  5. Repeat step 2-4 for 10 cycles  6. Final extension 72 degrees, 5 minute  7. Cool down 4 degrees, one hour</td><td>&nbsp;</td></tr> <tr><td>&nbsp;</td><td>PCR (Retry) / Gel</td><td>Kevin</td><td>&nbsp;</td><td>PCR Retry using XLI Blue:<br>Buffer 5 ul<br>DNTPs 1 ul<br>LP 1.5 ul<br>Rp 1.5 ul<br>Template 10 ul<br>Vent 0.7 ul<br>MgSO4 2 ul<br>H20 28.3 ul<br>Program:1. Denaturation (Initial): 95  for 5 mins 2. Denaturation: 98C for 20 seconds 3. Annealing: 54C for 15 seconds  4. Extension: 72 degrees, 30 seconds  5. Repeat step 2-4 for 36 cycles  6. Final extension 72 degrees, 5 minute  7. Cool down 4 degrees, one hour</td><td>Gel of FliC w/ from XLI Blue:<br>1) Ladder, 2) XL1 T1, 3) XL1 T2, 4) XL1 Control, 5) Ladder 6)XLIB 1, 7) XLIB 2, 8) XLIB 3<br></td></tr> <tr><td>&nbsp;</td><td>Miniprep</td><td>Kevin</td><td>B0015 - Double Terminator Trial 1, J04500 - T7 Promotoer (Two Trials), <br>B0011 - Weak RBS (Two Trials), BB030 - Strong RBS (Two Trials), 2 Trials of the rest of the parts from Jul-5 except double terminator 2 (B0017, only one trial)</td><td>&nbsp;</td><td>For next time, only use 1 mL of culture <br>so that there's less stuff after adding <br>solution 3.<br>Easier to pipette supernatent</td></tr></table></div>
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<p> <h1> Tuesday July 3rd </p> </h1>
<p> <h1> Tuesday July 3rd </p> </h1>
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To start the day, we examined the gels we ran on Friday to try to decide our next plan of attack for isolating fliC C1. Should we try isolating genomic DNA of BL21 again, perhaps using a different protocol that doesn’t involve spooling the DNA out with a Pasteur pipette? Or should we abandon BL21 altogether and try a different strain, such as HB101? It might be a good idea to set a deadline for isolating fliC C1, and just get the gene synthesized if we are unable to do so by that date. Throughout the day, we continued research in our respective sub-groups, and significant progress was made in the Cohesins and Dockerins group. In the lab, we tried some digestions and ligations of parts with fliC C2, and plated competent cells of BL21 DE3 pLysS, XL1 Blue, and DH5-alpha. After lunch, Kevin brought in mini Canada Day-themed cupcakes for everyone. </p> </h2>
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To start the day, we examined the gels we ran on Friday to try to decide our next plan of attack for isolating fliC C1. Should we try isolating genomic DNA of BL21 again, perhaps using a different protocol that doesn’t involve spooling the DNA out with a Pasteur pipette? Or should we abandon BL21 altogether and try a different strain, such as HB101? It might be a good idea to set a deadline for isolating fliC C1, and just get the gene synthesized if we are unable to do so by that date. Throughout the day, we continued research in our respective sub-groups, and significant progress was made in the Cohesins and Dockerins group. In the lab, we tried some digestions and ligations of parts with fliC C2, and plated competent cells of BL21 DE3 pLysS, XL1 Blue, and DH5-alpha. After lunch, Kevin brought in mini Canada Day-themed cupcakes for everyone. </h2></p>
<p> <h1>Wednesday July 4th</p> </h1>
<p> <h1>Wednesday July 4th</p> </h1>
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All the ligations from yesterday failed, unfortunately. In the lab this morning, Victor and David tried ligations of fmT and GFP with fliC C2. In the afternoon, Victor and Phillip grew liquid cultures for fliC C2 and the three different strains of E. coli that were plated yesterday. As for research topics, Kevin, Dan, and Andrew hunted for gene sequences for specific cohesins and dockerins. Phillip worked on haloacetate degradation and Beini worked on polymerization of flagellin subunits. </p> </h2>
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All the ligations from yesterday failed, unfortunately. In the lab this morning, Victor and David tried ligations of fmT and GFP with fliC C2. In the afternoon, Victor and Phillip grew liquid cultures for fliC C2 and the three different strains of E. coli that were plated yesterday. As for research topics, Kevin, Dan, and Andrew hunted for gene sequences for specific cohesins and dockerins. Phillip worked on haloacetate degradation and Beini worked on polymerization of flagellin subunits. </h2></p>
<p> <h1>Thursday July 5th</p> </h1>
<p> <h1>Thursday July 5th</p> </h1>
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Morning newsflash from Andrew: the incubator was set to 54°C. Our latest digestion-ligations didn’t work... now we don’t know whether it was because of the cranked up incubator or some other reason. For the future, we should either use the 37°C incubator room, or just be more careful about brushing the knob of the incubator.  
Morning newsflash from Andrew: the incubator was set to 54°C. Our latest digestion-ligations didn’t work... now we don’t know whether it was because of the cranked up incubator or some other reason. For the future, we should either use the 37°C incubator room, or just be more careful about brushing the knob of the incubator.  
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Our liquid cultures from yesterday worked, so we miniprepped those (fliC C2 + three strains of E. coli). We also attempted a second genomic DNA isolation, this time using a slightly different protocol that did not involve spooling the DNA out and transferring it to another tube. We did this for three strains of  E. coli (XL1 Blue, DH5-alpha, and BL21 DE3 pLysS).  
Our liquid cultures from yesterday worked, so we miniprepped those (fliC C2 + three strains of E. coli). We also attempted a second genomic DNA isolation, this time using a slightly different protocol that did not involve spooling the DNA out and transferring it to another tube. We did this for three strains of  E. coli (XL1 Blue, DH5-alpha, and BL21 DE3 pLysS).  
   
   
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Those involved in SynthetiQ met tonight and did some bodystorming for our upcoming PCR video, and well as some brainstorming about logistics. We decided that it would be ideal to have 8 dancers involved to effectively illustrate DNA concepts. </p> </h2>
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Those involved in SynthetiQ met tonight and did some bodystorming for our upcoming PCR video, and well as some brainstorming about logistics. We decided that it would be ideal to have 8 dancers involved to effectively illustrate DNA concepts. </h2></p>
<p> <h1>Friday July 6th </p> </h1>
<p> <h1>Friday July 6th </p> </h1>
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The lab was buzzing with activity today as we are in full swing with our digestions/ligations and PCRs. Victor, David, Andrew, and Phillip worked in the lab for most of the day, while Kevin and Beini researched the construction of fliD chimeras and motility assays. After lunch, Phillip brightened everyone’s day with chocolate chip cookies that he baked himself. They disappeared pretty quickly. Beini tried designing primers to amplify the fliD constant sequences, but the primers aren’t very ideal, so if we are planning to make fliD chimeras at all, we may want to consider getting the two constant sequences synthesized.  
The lab was buzzing with activity today as we are in full swing with our digestions/ligations and PCRs. Victor, David, Andrew, and Phillip worked in the lab for most of the day, while Kevin and Beini researched the construction of fliD chimeras and motility assays. After lunch, Phillip brightened everyone’s day with chocolate chip cookies that he baked himself. They disappeared pretty quickly. Beini tried designing primers to amplify the fliD constant sequences, but the primers aren’t very ideal, so if we are planning to make fliD chimeras at all, we may want to consider getting the two constant sequences synthesized.  
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Latest revision as of 22:17, 26 October 2012

Control

Notebook - Week 10

DateProtocolPeopleDNA (if relevant)Quantities and Parameters (if relevant)Notes on Protocol/ Gel Lanes
Jul-03Digestion and LigationDan, Victor, BeinipSB1C3 and GFP + fliC2 pSB1C3Digestion Mix: (GFP/fliC2)
Eco RI - HF / XbaI
SpeI / PstI
10X NEB Buffer 2
100X BSA
H20
 
 Streak Plates of competent cells BL21 DE3 plyss [C] 2 trials
XLI Blue [tet] 2 trials
DH5 - alpha [nal] 2 trials
  
 Heat shock transformationDan,FliC C2 in Kanamycin   
 Ligation DavidfmT PCR product into plasmid 1A3Ligation Mix:
2 ul PCR Product
1 ul EcoRI
1 ul PstI
5 ul NEBuffer 2
0.5 BSA
40.5 Water

Plasmid: 4 ul plasmid + 4 ul mastermix
 
 Heat shock transformationAndrew5 parts:
GFP + fliC C2 + psB1C3 (two trials)
GFP + pSB1C3
fliC2 + pSB1C3
fmt + pSB1A3
  
Jul-04Digestion and Ligation 1)GFP, 2) GFP, 3) GFP, 1) fmT, 2) fmT, 3) fmTDigestion Protocol (NEB):
NEB2: 2.5 ul
BSA: 0.5 ul
EcoRI: 0.5 ul
PstI : 0.5 ul

Used parts registry protocol for digestion:
1)GFP: DNA 10 ul, H20 6 ul, 2) GFP: DNA 5 ul, H20: 11 ul 3)GFP: DNA 2 ul, H20 14 ul, 1) fmT: DNA 10 ul, H20: 6 ul, 2)fmt: DNA 5 ul, H20 11 ul, 3) fmT: DNA 2 ul, H20 14 ul
 
 Liquid Cultures XLI Blue, BL21 DE5 Plys, DH5α, 2x fliC kanamycin,   
 Ligations 6 Ligations:
1) GFP + pSB1C3 ligation T1
2) GFP + pSB1C3 ligation T2
3) GFP + pSB1C3 ligation T3
4) fmt + pSB1A3 ligation T1
5) fmt + pSB1A3 ligation T2
6) fmt + pSB1A3 ligation T3
 Protocol was not recorded, NEB protocol was used.
Jul-05MiniprepVictor, Kevin, BeinifliC C2 x3   
 Genomic DNA Isolation XLI Blue, BL21 DE5 Plys, DH5α. 70% Ethanol required for the protocol
was diluted using 85% Ethanol.
 Primer Resuspension and
dilutions
DavidMetal-Binding Part primers:
Primer resuspension and dilutions for GoIB, SmtA and MBP
 note: MBP LP may not have exact amount of TE added during resuspension due to pipetting error.
Note: SmTA LP dilution may not have enough primer added --> If PCR failts try making another dilution
Jul-06PCRDavid, AndrewPbrR, SmtA, GolB
Four trials of each + one control each
Mastermix
Thermopol Rxn Buffer: 5 ul
DNTPs (10 mM): 1 ul
LP: 1.5 ul
RP: 1.5 ul
DNA Template: 5 ul
Vent Polymerase: 0.5 ul
MgSO4: 1 ul
H2O: 34.5 ul
Total: 50 ul
Program:1. Denaturation (Initial): 95 for 5 mins 2. Denaturation: 95C for 20 seconds 3. Annealing: 60C for 15 seconds 4. Extension: 72 degrees, 30 seconds 5. Repeat step 2-4 for 30 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour
Big Gel:
1) Ladder, 2)mCit FD digest EP, 3) PbrR Control
4) PbrR Trial 1, 5) PbrR Trial 2, 6) PbrR Trial 3, 7) PbrR Trial 4, 8) Ladder, 9) SmtA Control, 10) SmtA Trial 1, 11) SmtA Trial 2, 12) SmtA Trial 3, 13) SmtA Trial 4, 14) SmtA Trial 5, 15) Ladder, 16) GoIB Control, 17) GoIB Trial 1, 18) GoIB Trial 2, 19) GoIB Trial 3, 20) GoIB Trial 4, 21) GoIB Trial 5, 22) Ladder, 23) PbrR Trial 5.
 NEB Protocol DigestionVictorXLI Blue, BL21 DE5 Plys, DH5α, fliC C2, Luciferase, mCitrine,  Small Gel:
1) Ladder, 2) DH5α NEB EP Digest,3) DH5α Control, 4) XLI Control, 5) LI Digest Blue NEB EP, 6) BL21 Control, 7) BL21 Fast Digest EP, 8) fliC C3 Digest NEB EP, 9) Ladder,10) fliC C2 Control, 11) fliC C2 digest FD EP, 12) Luciferase Control, 13) Luciferase NEB T1 EP, 14) Luciferase NEB T2 EP, 15) mCitrine Control, 16) mCit FD Digest EP.
 Heat Shock TransformationAndrewParts list BBa_:
J04500 - Promoter, J13002 - Promoter, 1712074 - Promoter, B0010 - Terminator, B0015 - Terminator, B0017 - Terminator, B0030 - Strong RBS, B0031 - Weak RBS, B0032 - Medium RBS
  
Jul-06PCR (Thermopol Protocol)KevinFour trials of each strain + one controlThermopol Protocol:
Thermopol: 5 ul
dNTPs: 1 ul
LP: 1.5 ul
RP: 1.5 ul
DNA Template: 5 ul
Vent Pol: 0.5 ul
MgSO4: 1 ul
H2O: 34.5 ul

Program:1. Denaturation (Initial): 95 for 5 mins 2. Denaturation: 98C for 20 seconds 3. Annealing: 55C for 15 seconds 4. Extension: 72 degrees, 30 seconds 5. Repeat step 2-4 for 30 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour
 
Jul-07MiniprepKevin, MaggiefmT+ pSB1C3, GFP + pSB1C3 (2 trials each)  
 Digestions Using Trial #1 from above miniprepsNEB Digestion Protocol:
2 ul Templates
1 ul EcoRI HF
1 ul SpeI (NEB)
5 ul NEB Buffer 2
0.5 BSA
40.5 H20

Fast Digest Protocol:
2 ul Template
2 ul 10x FD Buffer Green
1 ul SpeI (FD)
1 ul EcoRI (NEB)
14 ul H20
 
 Gel Electrophoresis 28 Lanes total Large Gel:
1) Ladder, 2) Control (fliC), 3) XLI Blue T1
4) XLI Blue T2, 5) XLI Blue T3, 6) XLI Blue T4
7) Ladder, 8) DH5α T1, 9) DH5α T2, 10) DH5α T3, 11) DH5α T4, 12) Blank, 13) Blank, 14) Blank, 15) Ladder, 16) BL 21 T1, 17) BL 21 T2, 18) BL 21 T3, 19) BL 21 T4, 20) fmT mpp T1, 21) Ladder, 22) fmT NEB, 23) fmT FD (Green) 24) GFP Mpp, 28) GFP NEB, 29) GFP FD (Green)
Jul-08PCRKevin, MaggieXL1 BluePCR using XLI Blue:
Buffer 5 ul
DNTPs 1 ul
LP 1.5 ul
Rp 1.5 ul
Template 5 ul
Vent 0.5 ul
MgSO4 1 ul
H20 34.5 ul
Program:1. Denaturation (Initial): 95 for 5 mins 2. Denaturation: 98C for 20 seconds 3. Annealing: 55C for 15 seconds 4. Extension: 72 degrees, 30 seconds 5. Repeat step 2-4 for 10 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour
 
 PCR (Retry) / GelKevin PCR Retry using XLI Blue:
Buffer 5 ul
DNTPs 1 ul
LP 1.5 ul
Rp 1.5 ul
Template 10 ul
Vent 0.7 ul
MgSO4 2 ul
H20 28.3 ul
Program:1. Denaturation (Initial): 95 for 5 mins 2. Denaturation: 98C for 20 seconds 3. Annealing: 54C for 15 seconds 4. Extension: 72 degrees, 30 seconds 5. Repeat step 2-4 for 36 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour
Gel of FliC w/ from XLI Blue:
1) Ladder, 2) XL1 T1, 3) XL1 T2, 4) XL1 Control, 5) Ladder 6)XLIB 1, 7) XLIB 2, 8) XLIB 3
 MiniprepKevinB0015 - Double Terminator Trial 1, J04500 - T7 Promotoer (Two Trials),
B0011 - Weak RBS (Two Trials), BB030 - Strong RBS (Two Trials), 2 Trials of the rest of the parts from Jul-5 except double terminator 2 (B0017, only one trial)
 For next time, only use 1 mL of culture
so that there's less stuff after adding
solution 3.
Easier to pipette supernatent

Tuesday July 3rd

To start the day, we examined the gels we ran on Friday to try to decide our next plan of attack for isolating fliC C1. Should we try isolating genomic DNA of BL21 again, perhaps using a different protocol that doesn’t involve spooling the DNA out with a Pasteur pipette? Or should we abandon BL21 altogether and try a different strain, such as HB101? It might be a good idea to set a deadline for isolating fliC C1, and just get the gene synthesized if we are unable to do so by that date. Throughout the day, we continued research in our respective sub-groups, and significant progress was made in the Cohesins and Dockerins group. In the lab, we tried some digestions and ligations of parts with fliC C2, and plated competent cells of BL21 DE3 pLysS, XL1 Blue, and DH5-alpha. After lunch, Kevin brought in mini Canada Day-themed cupcakes for everyone.

Wednesday July 4th

All the ligations from yesterday failed, unfortunately. In the lab this morning, Victor and David tried ligations of fmT and GFP with fliC C2. In the afternoon, Victor and Phillip grew liquid cultures for fliC C2 and the three different strains of E. coli that were plated yesterday. As for research topics, Kevin, Dan, and Andrew hunted for gene sequences for specific cohesins and dockerins. Phillip worked on haloacetate degradation and Beini worked on polymerization of flagellin subunits.

Thursday July 5th

Morning newsflash from Andrew: the incubator was set to 54°C. Our latest digestion-ligations didn’t work... now we don’t know whether it was because of the cranked up incubator or some other reason. For the future, we should either use the 37°C incubator room, or just be more careful about brushing the knob of the incubator. A whole bunch of emails were sent today: contacting sponsors, ordering enzymes, requesting genes/plasmids from investigators, etc. Phillip updated the scroll to top button on our wiki. In the afternoon, we received a package from our sponsor Novus Biologicals, containing cool gear like antibody-shaped floaters in fun colours, highlighter-pens, lanyards, and fridge magnets. Our liquid cultures from yesterday worked, so we miniprepped those (fliC C2 + three strains of E. coli). We also attempted a second genomic DNA isolation, this time using a slightly different protocol that did not involve spooling the DNA out and transferring it to another tube. We did this for three strains of E. coli (XL1 Blue, DH5-alpha, and BL21 DE3 pLysS). Those involved in SynthetiQ met tonight and did some bodystorming for our upcoming PCR video, and well as some brainstorming about logistics. We decided that it would be ideal to have 8 dancers involved to effectively illustrate DNA concepts.

Friday July 6th

The lab was buzzing with activity today as we are in full swing with our digestions/ligations and PCRs. Victor, David, Andrew, and Phillip worked in the lab for most of the day, while Kevin and Beini researched the construction of fliD chimeras and motility assays. After lunch, Phillip brightened everyone’s day with chocolate chip cookies that he baked himself. They disappeared pretty quickly. Beini tried designing primers to amplify the fliD constant sequences, but the primers aren’t very ideal, so if we are planning to make fliD chimeras at all, we may want to consider getting the two constant sequences synthesized.