Difference between revisions of "Team:Missouri Rolla/Notebook"

m
m
 
(37 intermediate revisions by the same user not shown)
Line 117: Line 117:
 
   font-size: 13pt;
 
   font-size: 13pt;
 
   color: black;
 
   color: black;
−
   margin-bottom: 1em;
+
   margin-bottom: 0;
 +
}
 +
#mstigem table, tr, td {
 +
  font-family: 'Palatino Linotype', 'Book Antiqua', Palatino, serif;
 +
  font-size: 11pt;
 +
  border: 1px solid black;
 +
  border-spacing: 0;
 +
}
 +
#mstigem td {
 +
  padding: 2px;
 
}
 
}
 
#mstigem a {
 
#mstigem a {
Line 141: Line 150:
 
   margin-bottom: 0;
 
   margin-bottom: 0;
 
   position: relative;
 
   position: relative;
 +
}
 +
img.gel {
 +
  width:40%;
 
}
 
}
 
img.inlinephoto-left {
 
img.inlinephoto-left {
Line 193: Line 205:
 
     margin-left: 10vw;
 
     margin-left: 10vw;
 
     width: 80vw;
 
     width: 80vw;
 +
  }
 +
  img.gel {
 +
  width: 65%;
 
   }
 
   }
 
}
 
}
Line 199: Line 214:
 
     width: 46vw;
 
     width: 46vw;
 
     left: 15vw;
 
     left: 15vw;
 +
  }
 +
  img.gel {
 +
    width:100%;
 +
  }
 +
}
 +
@media screen and (max-width: 400px) {
 +
  #mstigem table, tr, td {
 +
    font-size: 9pt;
 +
  }
 +
}
 +
@media screen and (max-width: 335px) {
 +
  #mstigem table, tr, td {
 +
    font-size: 7.5pt;
 
   }
 
   }
 
}
 
}
Line 248: Line 276:
 
}
 
}
 
</style>
 
</style>
−
<a href="https://2015.igem.org/Team:Missouri_Rolla" onmousemove="mouse(event)"><img id="logo" src = "https://static.igem.org/mediawiki/2015/1/16/Mstigem_logo2015.png" /></a>
+
<a href="https://2015.igem.org/Team:Missouri_Rolla" ><img id="logo" src = "https://static.igem.org/mediawiki/2015/1/16/Mstigem_logo2015.png" /></a>
 
<img id="cave" src = "https://static.igem.org/mediawiki/2015/d/d1/Mstigem_cave_background.jpg" alt="Missouri cave photographed by Lynn Dieter" />
 
<img id="cave" src = "https://static.igem.org/mediawiki/2015/d/d1/Mstigem_cave_background.jpg" alt="Missouri cave photographed by Lynn Dieter" />
 
<div id="menu">
 
<div id="menu">
−
<a onmousemove="mouse(event)" href="https://2015.igem.org/Team:Missouri_Rolla">Project</a><a onmousemove="mouse(event)" href="https://2015.igem.org/Team:Missouri_Rolla/Practices">Human Practices</a><br class="menu2" /><a onmousemove="mouse(event)" href="https://2015.igem.org/Team:Missouri_Rolla/Parts">Parts</a><br class="menu1" /><a onmousemove="mouse(event)" href="https://2015.igem.org/Team:Missouri_Rolla/Notebook">Notebook</a><a onmousemove="mouse(event)" href="https://2015.igem.org/Team:Missouri_Rolla/Team">Team</a><br class="menu2" /><a onmousemove="mouse(event)" href="https://2015.igem.org/Team:Missouri_Rolla/Safety">Safety</a><a onmousemove="mouse(event)" href="https://2015.igem.org/Team:Missouri_Rolla/Attributions">Attributions</a>
+
<a href="https://2015.igem.org/Team:Missouri_Rolla">Project</a><a href="https://2015.igem.org/Team:Missouri_Rolla/Practices">Human Practices</a><br class="menu2" /><a href="https://2015.igem.org/Team:Missouri_Rolla/Parts">Parts</a><br class="menu1" /><a href="https://2015.igem.org/Team:Missouri_Rolla/Notebook">Notebook</a><a href="https://2015.igem.org/Team:Missouri_Rolla/Team">Team</a><br class="menu2" /><a href="https://2015.igem.org/Team:Missouri_Rolla/Safety">Safety</a><a href="https://2015.igem.org/Team:Missouri_Rolla/Attributions">Attributions</a>
 
</div>
 
</div>
−
<h1 onmousemove="mouse(event)">DEFENDING NORTH AMERICAN BATS FROM THE EMERGING WHITE-NOSE EPIDEMIC</h1>
+
<h1 >NOTEBOOK</h1>
−
<div onmousemove="mouse(event)" id="mstigem">
+
<div id="mstigem">
−
<p onmousemove="mouse(event)"><b>Date: 5/22/2015</b> <b>People in lab: Kent Gorday</b><br /><b>Electrical Transformation of 2015 iGEM Plate 1 3O</b><br /><b>Start Time:</b> 3:00pm<br /><b>Purpose:</b> To transform and clone a promoter and rbs in standard iGEM backbone for later addition of last year's hmp part.<br /><b>Protocol:</b> 1.5uL of BBa_K608002, previously taken from iGEM Plate 1 3O, was electrically transformed into competent cell. An 1.8kV pulse was applied according to Lab Manual procedure. Plates were left to incubate overnight.<br /><b>Products:</b></p><table onmousemove="mouse(event)"><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>5/22 ET1 KG 20µL</td><td>BBa_K608002 in E. coli on chloramphenicol, 20 µL plating volume</td><td>2015 iGEM Plate 1 3O</td></tr><tr><td>5/22 ET1 KG 200µL</td><td>BBa_K608002 in E. coli on chloramphenicol, 200 µL plating volume</td><td>2015 iGEM Plate 1 3O</td></tr></table><p onmousemove="mouse(event)"><b>Stop Time:</b> 4:10pm<br /><b>Next:</b> InocµLate liquid cµLture from 5/22 ET1 then miniprep product and glycerol stock of hmp.<br /></p>
+
<p>Many of our standard procedures can be found in our <a href="https://static.igem.org/mediawiki/2015/0/09/Mstigem_ltm_e2.pdf">Lab Training Manual</a>.</p>
 +
</div>
 +
<div  id="mstigem">
 +
<p ><b>Date: 5/22/2015<br />People in lab: Kent Gorday</b><br /><b>Electrical Transformation of 2015 iGEM Plate 1 3O</b><br /><b>Start Time:</b> 3:00pm<br /><b>Purpose:</b> To transform and clone a promoter and rbs in standard iGEM backbone for later addition of last year's hmp part.<br /><b>Protocol:</b> 1.5µL of BBa_K608002, previously taken from iGEM Plate 1 3O, was electrically transformed into competent cell. An 1.8kV pulse was applied according to Lab Manual procedure. Plates were left to incubate overnight.<br /><b>Products:</b></p><table ><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>5/22 ET1 KG 20µL</td><td>BBa_K608002 in E. coli on chloramphenicol, 20 µL plating volume</td><td>2015 iGEM Plate 1 3O</td></tr><tr><td>5/22 ET1 KG 200µL</td><td>BBa_K608002 in E. coli on chloramphenicol, 200 µL plating volume</td><td>2015 iGEM Plate 1 3O</td></tr></table><p ><b>Stop Time:</b> 4:10pm<br /><b>Next:</b> Inoculate liquid culture from 5/22 ET1 then miniprep product and glycerol stock of hmp.</p>
 +
</div>
 +
<div  id="mstigem">
 +
<p ><b>Date: 5/25/2015<br />People in lab: Kira Buckowing</b> <br /><b>InocµLations of hmp frozen stock and colonies from ET1 5/22 200µL plate</b><br /><b>Start Time:</b> 3:00pm<br /><b>Purpose:</b> To have liquid culture grown up of hmp and the iGEM kit parts (Promoter + rbs)<br /><b>Protocol:</b> Inoculation of 2 broth cultures from each the hmp frozen stock and the ET1 5/22 200µL plate. Wire loop was used for 3 of the 4 total samples, pipette for the last hmp broth culture. Procedure taken from iGEM LTP Lab Manual edition 2<br /><b>Products:</b></p><table ><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>BC1 KKB 5/25</td><td>iGEM kit part, liquid culture</td><td>ET1 5/22</td><td>2</td></tr><tr><td>BC3 KKB 5/25</td><td>hmp stock liquid culture</td><td>hmp glycerol stock</td><td>2</td></tr></table><p ><b>Stop Time:</b> 3:25pm<br /><b>Next:</b> Minipreps of all samples to isolate the desired plasmids.</p>
 +
</div>
 +
<div  id="mstigem">
 +
<p ><b>Date: 5/26/2015<br />People in lab: Kent Gorday</b><br /><b>Miniprep and digestion of hmp, pro/rbs, and amp BB</b><br /><b>Start Time:</b> 3:09pm<br /><b>Purpose:</b> To extract and digest BBa_K608002, hmp, and linearized pSB1A3<br /><b>Protocol:</b> Solution miniprep procedure attached to front of lab training manual was used on 3mL of liquid culture. Minipreps 1-4 were analyzed by nanodrop and showed no DNA due to a mistake in miniprep procedure. Identical minipreps were performed using the remaining 2mL of culture. Nanodrop then showed DNA presence, so the following digests were prepared: D1- 20.5 µL pSB1A3, 2.5 µL 10x Tango, 1 µL EcoRI, 1 µL PstI; D2 - 14.5 µL water, 2.5 µL 10x Tango, 6 µL MP5, 1 µL EcoRI, 1 µL SpeI; D3 - 18.5 µL water, 2.5 µL 10x Tango, 2 µL MP7, 1 µL XbaI, 1 µL PstI<br /><b>Products:</b></p><table ><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>KG 5/26 MP1, MP2, MP5, MP6</td><td>pro/rbs BBa_K68002</td><td>5/25 BC1, BC2</td><td>4</td></tr><tr><td>KG 5/26 MP3, MP4, MP7, MP8</td><td>hmp</td><td>5/25 BC1, BC2</td><td>4</td></tr><tr><td>KG 5/26 D1</td><td>E&P amp BB</td><td>Linear pSB1A3 2015</td><td>1</td></tr><tr><td>KG 5/26 D2</td><td>E&S pro/rbs</td><td>5/26 MP5</td><td>1</td></tr><tr><td>KG 5/26 D3</td><td>X&P hmp</td><td>5/26 MP7</td><td>1</td></tr></table><p ><b>Results:</b> MP1 - [DNA]: -4.9, 260/280: 1.65, 260/230: 0.06; MP2 - [DNA]: -2.2, 260/280: 1.28, 260/230: 0.04; MP3 - [DNA]: 13.8, 260/280: 1.38, 260/230: 0.35; MP4 - [DNA]: -4.8, 260/280: 1.65, 260/230: 0.08; MP5 - [DNA]: 161.8, 260/280: 1.91, 260/230: 2.51; MP6 - [DNA]: 150, 260/280: 1.87, 260/230: 2.28; MP7 - [DNA]: 508.5, 260/280: 1.85, 260/230: 1.84; MP8 - [DNA]: 462.9, 260/280: 1.82, 260/230: 1.55<br /><b>Stop Time:</b> 7:50pm<br /><b>Next:</b> Ligate 5/26 D1, D2, D3</p>
 +
</div>
 +
<div  id="mstigem">
 +
<p ><b>Date: 5/27/2015<br />People in lab: Kira Buckowing</b><br /><b>Ligation of hmp/pro/rbs/amp BB</b><br /><b>Start Time:</b> 3:15pm<br /><b>Purpose:</b> To combine the wanted digest parts from 5/26 into one plasmid (3A assembly)<br /><b>Protocol:</b> Ligation protocol from LM ed2 with the following adjustments: 1µL each of the hmp section and pro/rbs section used for L1 & 2µL each used for L2 with 4µL of the amp backbone<br /><b>Products:</b></p><table ><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>L1 KKB 5/27</td><td>1µL D2, 1 µL D3, 6µL D1; ligated plasmid</td><td>5/26 KG D1, D2, D3</td></tr><tr><td>L2 KKB 5/27</td><td>2µL D2, 1 µL D3, 6µL D1; ligated plasmid</td><td>5/26 KG D1, D2, D3</td></tr></table><p ><b>Stop Time:</b> 4:25PM<br /><b>Next:</b> Test plasmid with gel electrophoresis or go straight to transformation to see if the plasmid ligated as planned</p>
 +
</div>
 +
<div  id="mstigem">
 +
<p ><b>Date: 5/28/2015<br />People in lab: Kent Gorday</b><br /><b>Electrical transformation of pro/rbs+hmp+amp</b><br /><b>Start Time:</b> 3:00pm<br /><b>Purpose:</b> To transform functioning hmp plasmid into competent cells for cloning<br /><b>Protocol:</b> 2µL of the ligations from 5/27 were transformed into electrocompetent cells using a 1.8kV pµLse and LM procedure. Plates were left to incubate overnight.<br /><b>Products:</b></p><table ><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>5/28 ET1</td><td>hmp+pro/rbs on amp plate, no arc</td><td>5/27 L1</td><td>2</td></tr><tr><td>5/28 ET2</td><td>hmp+pro/rbs on amp plate, arced</td><td>5/27 L1</td><td>2</td></tr><tr><td>5/28 ET3</td><td>hmp+pro/rbs on amp plate, no arc</td><td>5/27 L2</td><td>2</td></tr><tr><td>5/28 ET3</td><td>hmp+pro/rbs on amp plate, arced</td><td>5/27 L2</td><td>2</td></tr></table><p ><b>Stop Time:</b> 4:15pm<br /><b>Next:</b> Inoculate liquid cultures from transformations then miniprep</p>
 +
</div>
 +
<div  id="mstigem">
 +
<p ><b>Date: 6/7/2015<br />People in lab: Kent Gorday</b><br /><b>Inoculation of liquid cultures amp+hmp+pro/rbs</b><br /><b>Start Time:</b> 4:25pm<br /><b>Purpose:</b> To clone hmp+pro/rbs/amp plasmid for later miniprep<br /><b>Protocol:</b> According to inoculation protocol, 5µL of ampicillin was added to each 5µL broth culture, then left to incubate overnight<br /><b>Products:</b></p><table ><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>6/7 KG BC1</td><td>1x amp, amp/pro/rbs</td><td>5/28 ET1 20µL</td><td>4</td></table><p ><b>Stop Time:</b> 4:55pm<br /><b>Next:</b> Miniprep cultures and digest</p>
 +
</div>
 +
<div  id="mstigem">
 +
<p ><b>Date: 6/8/2015<br />People in lab: Kent Gorday</b><br /><b>Observation</b><br /><b>Start Time:</b> 2:20pm<br /><b>Purpose:</b> Observe broth cultures<br /><b>Notes:</b> Cultures from 6/7 show only slight growth and were left to incubate again overnight. Preparation of miniprep solutions was completed and solutions put in refrigerator, <br /><b>Stop Time:</b> 3:35pm<br /><b>Next:</b> Miniprep cultures and digest</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 6/9/2015<br />People in lab: Kent Gorday</b><br /><b>Inoculation of liquid cultures</b><br /><b>Start Time:</b> 11:25am<br /><b>Purpose:</b> To clone hmp/pro/rbs+amp for later miniprep<br /><b>Protocol:</b> 5µL of amp was added to each 5mL LB tube, then a sterile pipette tip used to inoculate the cultures near a flame. Cultures were left in shaking incubator.<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>KG 6/9 BC1</td><td>1x amp, hmp+pro+rbs</td><td>KG 5/28 ET2 20µL</td><td>2</td></tr></table><p><b>Stop Time:</b> 11:40am<br /><b>Next:</b> Miniprep cultures and digest</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 6/10/2015<br />People in lab: Kent Gorday</b><br /><b>Miniprep, digest, ligation of hmp/pro/rbs</b><br /><b>Start Time:</b> 12:50pm<br /><b>Purpose:</b> To produce a completed pro/rbs/hmp part for submission<br /><b>Protocol:</b> Three solution minipreps were performed using 1.5mL of 6/7 BC3 and BC4, and 3mL of 6/9BC1. The amounts of solutions 1,2,3 used per miniprep were 150, 200, and 150µL, respectively. DNA was resuspended in 30µL of TE buffer. Digests: D1 - 10.5µL milliQ water, 2.5µL Tango, 1µL EcoRI, 1 µL PstI, 10µL 6/10MP1; D2 - 3.5µL MilliQ, 2.5µL Tango, 1 µL EcoRI, 1 µL PstI, 17µL 6/10 MP3; D3 - 2.5µL Tango, 1µL EcoRI, 1µL PstI, 20.5 µL pSB1C3 2015. Ligations: L1 - 6µL D3, 2µL D1,1µL 10x T4 buffer, 1µL T4 ligase; L2 - 6µL D3, 2µL D2, 1µL 10x buffer, 1µL T4 ligase; L3 - 4 µL D3, 4µL D1, 1µL 10x buffer, 1µL T4 ligase; L4 - 4µL D3, 4µL D2, 1µL 10x buffer, 1µL T4 ligase<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>KG 6/10 MP1,2</td><td>hmp+pro/rbs+amp</td><td>6/7 BC3, 4</td><td>2</td></tr><tr><td>KG 6/10 MP3</td><td>hmp+pro/rbs+amp</td><td>6/9 BC1</td><td>1</td></tr><tr><td>6/10 KG D1, 2</td><td>pro/rbs+hmp cut E&P</td><td>6/10 KG MP1, 3</td><td>2</td></tr><tr><td>6/10 D3 KG</td><td>chlorBB cut E&P</td><td>pSB1C3 2015</td><td>1</td></tr><tr><td>6/10 KG L1, 2, 3, 4</td><td>completed hmp+pro/rbs part for igem</td><td>6/10 D3&D1, D2&D3, D3&D1, D3&D2</td><td>4</td></tr></table><p><b>Results:</b> MP1 - [DNA]: 100, 260/280: 1.82, 260/230: 1.84; MP2 - [DNA]: 98.5, 260/280: 1.81, 260/230: 1.93; MP3 - [DNA]: 60.7, 260/280: 1.84; 260/230: 2.35<br /><b>Stop Time:</b> 5:00pm<br /><b>Next:</b> Transform ligations to clone and miniprep</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 6/11/2015<br />People in lab: Kira Buckowing, Kent Gorday</b><br /><b>Electrical transformation of hmp/rbs part and 2J from kit</b><br /><b>Start Time:</b> 4:40pm<br /><b>Purpose:</b> To grow colonies with the wanted plasmids<br /><b>Protocol:</b> 2J DNA resuspended in 10µL milliQ, ETs per lab manual<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>2J KKB 6/11/15</td><td>Part BBa_J04450 in pSB4A5 backbone, rfp in amp</td><td>iGEM DNA Plate 4 well 2J</td><td>1</td></tr><tr><td>6F KKB 6/11/15</td><td>Part BBa_J04450 in pSB3K3 bb, rfp in kan</td><td>iGEM DNA plate 4 well 6F</td><td>1</td></tr><tr><td>KG 6/11 ET1, ET2, ET3, ET4</td><td>ET of L1, L1, L2, L2, L3 L3</td><td>KG 6/10 L1, L1, L2, L2, L3, L3</td><td>8</td></tr><tr><td>ET5 KKB 6/11</td><td>2J Kit part ET</td><td>ET5 of 2J</td><td>2</td></tr><tr><td>ET6 KKB 6/11</td><td>6F Kit part ET</td><td>ET6 of 6F</td><td>2</td></tr></table><p><b>Notes:</b> ET6 arced. No growth -KKB 6/13<br /><b>Stop Time:</b> 6:20pm<br /><b>Next:</b> Check for growth</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 6/16/2015<br />People in lab: Kent Gorday</b><br /><b>New electrical transformations of hmp/pro/rbs and 2J, 6F kit parts</b><br /><b>Start Time:</b> 4:56 pm<br /><b>Purpose:</b> To transform parts fro cloning<br /><b>Protocol:</b> LM ed. 2, D1 through D4 - 17.5µL milliQ, 2.5 Tango, 1µL PstI, and 4µL of 6/10 L1 through L4, respectively.<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>6/16 D1, 2, 3, 4</td><td>hmp/pro/rbs digested with P</td><td>KG 6/10 L1, L2, L3, L4</td><td>4</td></tr><tr><td>6/16 ET1, 2</td><td>hmp/pro/rbs on chlor, plated in duplicates 20 and 200µL</td><td>KG 6/10 L1, L2</td><td>4</td></tr><tr><td>6/16 ET3</td><td>J04450 on amp, plated in duplicates 20/200µL</td><td>KKB 6/11 2J</td><td>2</td></tr><tr><td>6/16 ET4</td><td>J04450 on kan, plated in duplicates 20/200µL</td><td>KKB 6/11 6F</td><td>2</td></tr></table><p><b>Stop Time:</b> 6:35pm<br /><b>Next:</b> If hmp plates grow, prepare part for iGEM submission. Otherwise run gel electrophoresis of digests to diagnose.</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 6/17/2015<br />People in lab: Kent Gorday</b><br /><b>Gel electrophoresis of hmp/pro/rbs digested with PstI</b><br /><b>Start Time:</b> 4:50pm<br /><b>Purpose:</b> To troubleshoot hmp/pro/rbs using gel electrophoresis<br /><b>Protocol:</b> LM ed2<br /><b>Products:</b></p><table><tr><td>Well</td><td>Sample</td></tr><tr><td>3</td><td>5µL low range ladder</td></tr><tr><td>4</td><td>6/16 D1 15µL</td></tr><tr><td>5</td><td>6/16 D2 15µL</td></tr><tr><td>6</td><td>6/16 D3 15µL</td></tr><tr><td>7</td><td>6/16 D4 15µL</td></tr></table><p><b>Results:</b><br /><img class="gel" src="https://static.igem.org/mediawiki/2015/c/cd/Mstigem_jun17.jpeg" alt="gel electrophoresis" /><br /><b>Notes:</b> Calculated fragment lengths for all ligations: 2300bp, 4000bp<br /><b>Stop Time:</b> 6:48pm<br /><b>Next:</b> Run gel of MP 5/26 and digests from 6/10</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 6/18/2015<br />People in lab: Kent Gorday</b><br /><b>Gel electrophoresis of 6/10 digests and 5/26 MP</b><br /><b>Start Time:</b> 7:20pm<br /><b>Purpose:</b> To continue troubleshooting hmp/pro/rbs using gel electrophoresis<br /><b>Protocol:</b> LM ed2, Digests: D1 - 11.5 MilliQ, 2.5µL Tango, 10µL 5/26 P1, 1µL PstI<br /><b>Products:</b></p><table>    <tr>
 +
        <td>Well</td>
 +
        <td>Sample</td>
 +
    </tr>
 +
    <tr>
 +
        <td>2</td>
 +
        <td>5µL low range ladder</td>
 +
    </tr>
 +
    <tr>
 +
        <td>3</td>
 +
        <td>6/16 D1, 10µL</td>
 +
    </tr>
 +
    <tr>
 +
        <td>4</td>
 +
        <td>6/18 D1, 15µL</td>
 +
    </tr>
 +
    <tr>
 +
        <td>5</td>
 +
        <td>6/10 D1, 10µL</td>
 +
    </tr>
 +
    <tr>
 +
        <td>6</td>
 +
        <td>6/10 D3 15µL</td>
 +
    </tr></table><p><b>Stop Time:</b> 11:00pm<br /><b>Next:</b> Repeat transformation of 5/27 L1 & 2 with new ampicillin plates</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 6/19/2015<br />People in lab: Kent Gorday</b><br /><b>Gel purification and ligation of hmp/pro/rbs</b><br /><b>Start Time:</b> 6:00pm<br /><b>Purpose:</b> To assemble pro/rbs/hmp using gel purification<br /><b>Protocol:</b> LM ed2, Digest D1 - 20.5 µL 2015 pSB1C3, 2.5 µL Tango, 1µL EcoRI, 1µL PstI, Ligation: L1 - 4µL 6/19 GE1, 2µL GE2, 2µL GE3, 1µL buffer, 1µL T4 ligase<br /><b>Products:</b></p><table><tr><td>Well</td><td>Sample</td></tr><tr><td>1</td><td>6/19 D1, 15µL</td></tr><tr><td>2</td><td>5/26 D2, 15µL</td></tr><tr><td>3</td><td>5/26 D3, 15µL</td></tr><tr><td>4</td><td>Low range ladder 5µL</td></tr></table><p><b>Results:</b> Nanodrop showed no DNA in gel purifications.<br /><b>Stop Time:</b> 10:45pm<br /><b>Next:</b> Repeat transformation of 5/27 L1 & 2 on new ampicillin plates</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 6/20/2015<br />People in lab: Kent Gorday</b><br /><b>Electrical transformations of hmp+pro/rbs on amp/chlor</b><br /><b>Start Time:</b> 2:52pm<br /><b>Purpose:</b> to clone hmp+pro/rbs plasmids<br /><b>Protocol:</b> 1µL of ligations transformed into ecomp cells<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>6/20 ET1</td><td>hmp/pro/rbs on amp</td><td>5/27 L1</td><td>2</td></tr><tr><td>6/20 ET2</td><td>hmp/pro/rbs on amp</td><td>5/27 L2</td><td>2</td></tr><tr><td>6/20 ET3</td><td>hmp/pro/rbs on chlor</td><td>6/20 L1</td><td>2</td></tr></table><p><b>Stop Time:</b> 4:00pm<br /><b>Next:</b> Inoculate liquid cultures and miniprep</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 6/22/2015<br />People in lab: Kent Gorday</b><br /><b>Inoculation of liquid cultures of hmp/pro/rbs/amp</b><br /><b>Start Time:</b> 9:52am<br /><b>Purpose:</b> To clone plasmid for later miniprep<br /><b>Protocol:</b> 5µL amp added to tubes, inoculated with sterile pipette tips<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>6/22 BC1</td><td></td><td>6/20 ET2</td><td>2</td></tr></table><p><b>Stop Time:</b> 10:10am<br /><b>Next:</b> Miniprep and possibly find different amp plates</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 6/22/2015<br />People in lab: Kira Buckowing</b><br /><b>ET of 2J and hmp/amp</b><br /><b>Start Time:</b> 4:35 pm<br /><b>Purpose:</b> To transform wanted plasmids into ecoli for later use<br /><b>Protocol:</b> LM ed2<br /><b>Exceptions:</b> 2µL each of hmp used<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>ET1 6/22 KKB</td><td>2J kit parts, RFP</td><td>2J KKB</td><td>2</td></tr><tr><td>ET2 6/22 KKB</td><td>hmp/pro/rbs in amp</td><td>hmp L1 5/27</td><td>2</td></tr><tr><td>ET3 6/22 KKB</td><td>hmp/pro/rbs in amp</td><td>hmp L2 5/27</td><td>2</td></tr></table><p><b>Notes:</b> ET3 popped, ET2 was spilled<br /><b>Stop Time:</b> 6:35pm<br /><b>Next:</b> Inoculation of broth cultures</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 6/23/2015<br />People in lab: Kira Buckowing</b><br /><b>Inoculation of pro/rbs/hmp, 2J, 6F kit parts</b><br /><b>Start Time:</b> 4:45pm<br /><b>Purpose:</b> To obtain liquid cultures for minipreps<br /><b>Protocol:</b> LM ed2<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Souce Label</td><td>Quantity</td></tr><tr><td>ET3 BC1 KKB</td><td></td><td>ET3 6/22</td><td>1</td></tr><tr><td>ET BC2 KKB</td><td>1</td><td>ET2 6/22</td><td>1</td></tr><tr><td>ET1C BC3, 4 KKB</td><td></td><td>colony from lid of dish 2J</td><td>2</td></tr><tr><td>BC5, 6 KKB</td><td></td><td>colony of 6F</td><td>2</td></tr></table><p><b>Stop Time:</b> 5pm<br /><b>Next:</b> miniprep</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 6/24/2015<br />People in lab: Kent Gorday</b><br /><b>Start Time:</b> 7:55pm<br /><b>Purpose:</b> To assemble pro/rbs/hmp/chlor and ensure identity of product<br /><b>Protocol:</b> Kitless minipreps procedure. Digests: D1 - 18.5 milliq, 2.5 tango, 2 6/24 MP1, 1 EcoRI, 1 PstI; D2 - 10.5 milliq, 2.5 tango, 10 6/24 MP3, 1µL EcoRI, 1µL PstI; D3 - 10.5milliq, 2.5 tango, 10 pSB1C3 2014, 1µL EcoRI, 1µL PstI. Ligation: L1 - 5µL D3, 3µL D2, 1µL buffer, 1µL ligase<br /><b>Exceptions:</b> 150µL of soln 1, 200 µL of soln 2, 175µL of sln 3, 550µL isopropyl alcohol.<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Souce Label</td><td>Quantity</td></tr><tr><td>6/24 MP1</td><td>2J plate 4, lid colony</td><td> 6/23 BC4ET1C</td><td>1</td></tr><tr><td>6/24 MP2, 3</td><td>hmp/pro/rbs amp</td><td> 6/22 BC1, 2</td><td>2</td></tr><tr><td>6/24 ET1, 2</td><td>hmp/pro/rbs/amp</td><td>5/27 L1,2</td><td>4</td></tr><tr><td>6/24 D1</td><td>2J digested E&P</td><td> 6/24 MP1</td><td>1</td></tr><tr><td>6/24 D2</td><td>hmp/pro/rbs digested E&P</td><td>6/24 MP3</td><td>1</td></tr></table><p><b>Results:</b> MP1 - [DNA]: 524.4ng/µL, 260/280: 1.85, 260/230: 2.32; MP3 - [DNA]: 96/73, 260/280: 1.82, 260/230: 1.75<br /><b>Notes:</b> 6/24 D3,pSB1C3 2014, linear B digested E&P; 6/24 L1, 6/24 D2&D3, hmp/pro/rbs chlor<br /><b>Stop Time:</b> 12:01am<br /><b>Next:</b> Run gel of digests and transform ligations if lengths are correct.</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 6/25/2015<br />People in lab: Kent Gorday</b><br /><b>Gel elecetrophoresis of MPs</b><br /><b>Start Time:</b> 4:30pm<br /><b>Purpose:</b> to ensure identity of miniprep products<br /><b>Protocol:</b> LM ed2<br /><b>Products:</b></p><table><tr><td>Well</td><td>Sample</td></tr><tr><td>3</td><td>5µL low range ladder</td></tr><tr><td>4</td><td>10µL 6/24 D1</td></tr><tr><td>5</td><td>10µL 6/24 D2</td></tr></table><p><b>Stop Time:</b> 5:40pm<br /><b>Next:</b> religate hmp/pro/rbs amp</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 6/27/2015<br />People in lab: Kent Gorday</b><br /><b>New ligation of hmp/pro/rbs+amp</b><br /><b>Start Time:</b> 8:55pm<br /><b>Purpose:</b> To porform new ligations of 5/26 D1, 2, and 3 since gel electrophoresis showed failed of 5/27 ligations<br /><b>Protocol:</b> the following ligations were prepared and left to react overnight, L1 - 5/26 D1 7µL, 0.8µL D3, 0.2µL D2, 1µL 10x buffer, 1µL T4 ligase <br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>6/27 L1</td><td>hmp/pro/rbs + amp</td><td>5/26 D1, D2, D3</td></tr></table><p><b>Stop Time:</b> 9:35 pm<br /><b>Next:</b> Transform ligation, culture, and miniprep</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 6/28/2015<br />People in lab: Kent Gorday</b><br /><b>Electrical transformation of kit parts and hmp/pro/rbs/amp</b><br /><b>Start Time:</b> 6:05pm<br /><b>Purpose:</b> to clone plasmids for later inoculation and miniprep<br /><b>Protocol:</b> 10µL MilliQ water was used to resuspend 2014 plate 4 6F. Four electrical transformations were prepared according to the lab manual.<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>6/28 ET1, 2</td><td>hmp/pro/rbs/amp</td><td>2µL 6/27 L1</td><td>2</td></tr><tr><td>6/28 ET3</td><td>2J on amp</td><td>1µL 2015-4-2J</td><td>1</td></tr><tr><td>6/28 ET4</td><td>6F on kan</td><td>1µL 2014-4-6F</td><td>1</td></tr></table><p><b>Stop Time:</b> 7:30pm<br /><b>Next:</b> Inoculate liquid cultures</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 6/30/2015<br />People in lab: Kira Buckowing</b><br /><b>gBlock DNA resuspension and restriction digest</b><br /><b>Start Time:</b> 4:40pm<br /><b>Purpose:</b> To get gBlock DNA into a usable form to prep the pSB1C3 backbone for project work.<br /><b>Protocol:</b> Digest as per Lab Manual and resuspensions as per IDT instructions: 1. Centrifuge tube for a few seconds 2. add TE up to 10 ng/µL 3. Vortex briefly 4. Incubate @ 50C for 20 min 5. Vortex and centrifuge again briefly 6. [Added step] transfer total to pcr tube<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>pSB1C3 Digested 6/30</td><td>pSB1C3 cut with E&P</td><td>pSB1C3 kit DNA</td><td>1</td></tr><tr><td>6/30/15 KKB 1-1, 1-2, 1-3, 1-4</td><td>10ng/µL DNA</td><td> Part 1 1of4, 2of4, 3of4, 4of4</td><td>4</td></tr><tr><td>6/30/15 KKB, 2.1-1,2.1-2,2.1-3,2.1-4</td><td>10ng/µL DNA</td><td> Part 2-1 1of4, 2of4, 3of4, 4of4</td><td>4</td></tr><tr><td>6/30/15 KKB, 2.2-1,2.2-2,2.2-3,2.2-4</td><td>10ng/µL DNA</td><td>Part 2-2 1of4, 2of4, 3of4, 4of4</td><td>4</td></tr><tr><td>Part3 6/30/15 KKB</td><td>10ng/µL DNA</td><td>Part 3 ocimene synthase</td><td>1</td></tr></table><p><b>Notes:</b> 100µL added to 2.2-1 instead of the calculated 50µL so the concentration is halfed from the expected. 2014 linearized pSB1C3 used.<br /><b>Stop Time:</b> 7:00pm<br /><b>Next:</b> Assembly of the gBlock DNA fragments</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 6/30/2015<br />People in lab: Kent Gorday</b><br /><b>Electrical transformation of Kit parts and hmp/pro/rbs+amp</b><br /><b>Start Time:</b> 8:30pm<br /><b>Purpose:</b> to clone plasmids for later inoculation and miniprep<br /><b>Protocol:</b> LM ed2<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>KG 6/30 ET1</td><td>hmp/pro/rbs on amp</td><td>6/27 L1</td><td>2</td></tr><tr><td>KG 6/30 ET2</td><td>pSB4A5 on amp</td><td> 2015 plate4 2J</td><td>2</td></tr><tr>KG 6/30 ET3</td><td>pSB3K3 on kan</td><td>2014 plate4 6F</td><td>1</td><td>2</td></tr></table><p><b>Stop Time:</b> 9:40pm<br /><b>Next:</b> inoculate broth cultures and miniprep</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/2/2015<br />People in lab: Kent Gorday</b><br /><b>Chemical transformations ond PCR of hmp for gst tagging</b><br /><b>Start Time:</b> 8:27pm<br /><b>Purpose:</b> clone plasmids and prepare hmp for gst tagging<br /><b>Protocol:</b> LM ed2, PCR - A1: 12.5 µL Taq MM, 11.3µL MilliQ, 0.2µL 5/26 MP7, 0.5 µL F hmp, 0.5 µL R hmp, Ligation: L1 - 1µL 10x buffer, 5.1 µL 5/26 D1, 2.4 µL 5/26 D2, 0.5 µL 5/26 D3, 1µL ligase<br /><b>Exceptions:</b> PCR Program: 94C - 2mins, 32 cycles of (94C - 26s, 55C- 40s, 68C - 3min), 68C - 7min, 4C - hold<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>7/2 BC1</td><td>2014 6F in kan</td><td>6/30 ET3</td><td>2</td></tr><tr><td>7/2 BC3</td><td>hmp in pSB1C3</td><td>hmp glycerol stock</td><td>1</td></tr><tr><td>7/2 A1</td><td>hmp prepaged for gst tagging</td><td>5/26 MP7</td><td>1</td></tr><tr><td>7/2 CT1</td><td>hmp/pro/rbs/amp</td><td>6/27 L1</td><td>1</td></tr><tr><td>2015 plate 4 well 2J</td><td>2015 2J plate4</td><td>1</td><td>1</td></tr></table><p><b>Notes:</b> 7/2 L1, 5/26 D1. D2, D3, hmp/pro/rbs/amp<br /><b>Stop Time:</b> 1:11am<br /><b>Next:</b> Run a gel of A1, 7/2 L1, 6/27 L1, miniprep BCs incolµLate new BCs from Cts</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/3/2015<br />People in lab: Kent Gorday</b><br /><b>Miniprep hmp and pSB3K3</b><br /><b>Start Time:</b> 3:30pm<br /><b>Purpose:</b> Obtain plasmids from culture<br /><b>Protocol:</b> Three minipreps were performed using the kitless miniprep protocol<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>7/3 MP1</td><td>pSB3K3 in TE</td><td>7/2 BC1</td></tr><tr><td>7/3 MP2</td><td>pSB3K3 in TE</td><td>7/2 BC2</td></tr><tr><td>7/3 MP3</td><td>hmp in pSB1C3</td><td>7/2 BC3</td></tr></table><p><b>Results:</b> MP2 - [DNA]: 320.1 ng/µL, 260/280: 1.89, 260/230: 2.38; MP3 - 216.4, 1.75, 1.8<br /><b>Stop Time:</b> 5:00pm<br /><b>Next:</b> ET of hmp/pro/rbs</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/3/2015<br />People in lab: Kent Gorday</b><br /><b>Electrical transformation of hmp/pro/rbs/amp</b><br /><b>Start Time:</b> 5:10pm<br /><b>Purpose:</b> assemble hmp in pSB1C3 <br /><b>Protocol:</b> LM Ed2<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>7/3 ET1</td><td>hmp/pro/rbs amp</td><td>7/2 L1</td><td>2</td></tr></table><p><b>Stop Time:</b> 6:22pm<br /><b>Next:</b> If no growth, run a new digestion of pSB1A3 and repeat 7/2 L1 using new BB. Inoculate BC and MPs from 7/2 CT2, then digest pSB3K3 and pSB4A5 to check by gel</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/4/2015<br />People in lab: Kent Gorday</b><br /><b>Inoculation of liquid cultures for pSB4A5</b><br /><b>Start Time:</b> 10:14 am<br /><b>Purpose:</b> clone pSB4A5 for later miniprep<br /><b>Protocol:</b> Two broth cultures were inoculated using a wire loop<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>7/4 BC1</td><td>RFP in pSB4A5</td><td>7/2 CT2</td><td>2</td></tr></table><p><b>Stop Time:</b> 10:22am<br /><b>Next:</b> Miniprep broth cultures</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/5/2015<br />People in lab: Kent Gorday</b><br /><b>Miniprep and digestion of pSB4A5, pSB3K3, pSB1A3</b><br /><b>Start Time:</b> 7:35pm<br /><b>Purpose:</b> prepare pSB4A5 and pSB3K3 for gel and use<br /><b>Protocol:</b> Kitless miniprep protocol and LM ed2, Digests: D1 - 1µL E, 1µL P, 2.5µL tango, 11.5 water, 9µL 7/3 MP2; D2 - 1 E, 1 P, 2.5 buffer, 20.5 7/5 MP2; D3 - 1 E, 1 P, 2.5 buffer, 20.4 pSB1A3<br /><b>Products:</b></p><table><tr><td>Sample  Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>7/5 MP1</td><td>RFP in pSB4A5</td><td>7/4 BC1</td><td>1</td></tr><tr><td>7/5 MP2</td><td>RFP in pSB4A5</td><td>7/4 BC2</td><td>1</td></tr><tr><td>7/5 D1, D2</td><td>RFP in pSB3K3 cut E&P</td><td>7/3 MP2</td><td>2</td></tr><tr><td>7/5 D3</td><td>pSB1A3 cut E&P</td><td>2015 Linear pSB1A3</td><td>1</td></tr></table><p><b>Results:</b> MP1 - [DNA]: 69.37, 260/280: 1.87, 260/230: 1.92; MP2 - 138.52, 1.71, 1.46<br /><b>Stop Time:</b> 10:30pm<br /><b>Next:</b> Run gel of digests, repeat 7/2 L1 using 7/5 D3</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/6/2015<br />People in lab: Kent Gorday</b><br /><b>Ligation of pro/rbs/hmp amp</b><br /><b>Start Time:</b> 9:51am<br /><b>Purpose:</b> assemble hmp/pro/rbs part<br /><b>Protocol:</b> L1 - 1µL ligase, 1µL buffer, 1.4 µL D3, 5µL D2, 1.6µL D3<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>7/6 L1</td><td>hmp/pro/rbs/amp</td><td>7/5 D3, 5/26 D2, 5/26 D3</td></tr></table><p><b>Stop Time:</b> 10:10am<br /><b>Next:</b> Run gel of 7/5 digests, transform ligation</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/6/2015<br />People in lab: Kira Buckowing</b><br /><b>HiFi assembly Part 1</b><br /><b>Start Time:</b> 10:15am<br /><b>Purpose:</b> To assemble part 1<br /><b>Protocol:</b> Following mixed in PCR tube and heated to 50C for 1 hr, HiFi - HiFi Part 1: 1.3µL pSB1C3, 1-1 2.6µL, 1-2 3.1µL, 1-3 3.1µL, 1-4 3.6µL, HiFi MM 13.2<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>HiFi part 1 7/6 KKB</td><td>Assembled Part 1 for project</td><td>pSB1C3 6/30, 1-1, 1-2, 1-3, 1-4 6/30</td></tr></table><p><b>Notes:</b> Slightly less than an hour incubation<br /><b>Stop Time:</b> 11:25pm<br /><b>Next:</b> Tramsform and gel/digest to check product</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/6/2015<br />People in lab: Kent Gorday</b><br /><b>Gel of backbone digests</b><br /><b>Start Time:</b> 11:28am<br /><b>Purpose:</b> To check identity of digests<br /><b>Protocol:</b> LM ed2<br /><b>Products:</b></p><table><tr><td>Well</td><td>Sample</td></tr><tr><td>3</td><td>Ladder 5µL</td></tr><tr><td>4</td><td>7/5 D1 10µL</td></tr><tr><td>5</td><td>7/5 D2 10µL</td></tr><tr><td>6</td><td>7/5 D3 10µL</td></tr></table><p><b>Stop Time:</b> 12:55pm<br /><b>Next:</b> Transform 7/6 L1</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/6/2015<br />People in lab: Kira Buckowing</b><br /><b>HiFi Assembly Part 2-1</b><br /><b>Start Time:</b> 1pm<br /><b>Purpose:</b> To assemble the Part 2-1 <br /><b>Protocol:</b> The following were mixed in a PCR tube and incubated @ 50C for an hour: pSB1C3 1.3µL, 2.1-1 4.6µL, 2.1-2 2.8µL, 2.1-3 2.8µL, 2.1-4 3.1µL, HiFi MM 14.6 µL<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>HiFi Part 2.1 7/6 KKB</td><td>Part 2-1 for project</td><td>pSB1C3 digested 6/30, 2.1-1, 2.1-2, 2.1-3, 2.1-4 6/30</td></tr></table><p><b>Stop Time:</b> 2:20pm<br /><b>Next:</b> Transformation into e. coli to check products</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/6/2015<br />People in lab: Kira Buckowing</b><br /><b>Assembly of part 2-2</b><br /><b>Start Time:</b> 4:30pm<br /><b>Purpose:</b> To assemble the 2-2 part<br /><b>Protocol:</b> The following were mixed in a PCR tube and incubated at 50C for an hour. HiFi assembly: Part 2.2 - pSB1C3 1.3µL, 2.2-1 2.8µL, 2.2-2 2.8µL, 2.2-3 2.8µL, 2.2-4 3.1µL, HiFi MM 12.8µL<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>HiFi Part 2.2 7/6 KKB</td><td> Part 2-2 for project work</td><td> pSB1C3 6/30 2.2-1, 2, 3,4 6/30</td></tr></table><p><b>Stop Time:</b> 5:50pm<br /><b>Next:</b> Transformation and gel to check for product correctness</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/6/2015<br />People in lab: Kent Gorday</b><br /><b>CT of HiFi assemblies and hmp/pro/rbs</b><br /><b>Start Time:</b> 7:10pm<br /><b>Purpose:</b> Transform HiFi assemblies to clone and verify plasmids and assembled hmp/pro/rbs part<br /><b>Protocol:</b> LM ed2<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>7/6 BC1, 2, 3</td><td>FP pSB4A5</td><td>7/2 CT2</td><td>3</td></tr><tr><td>7/6 ET1</td><td>hmp/pro/rbs</td><td>7/6 L1</td><td>2</td></tr><tr><td>7/6 ET2</td><td>part 1 on chlor</td><td>HiFi part 1 7/6</td><td>2</td></tr><tr><td>7/6 ET3</td><td>part 2-1 on chlor</td><td>HiFi part 2-1 7/6</td><td>2</td></tr><tr><td>7/6 ET4</td><td>Part 2-2 on chlor</td><td>HiFi part 2-2 7/6</td><td>2</td></tr></table><p><b>Stop Time:</b> 10:00 pm<br /><b>Next:</b> pSB4A5 and inoculte BC from transformations</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/7/2015<br />People in lab: Kira Buckowing</b><br /><b>Digest and gel of HiFi  assemblies</b><br /><b>Start Time:</b> 5:15pm<br /><b>Purpose:</b> To check validity of products<br /><b>Protocol:</b> LM ed 2<br /><b>Products:</b></p><table><tr><td>Well</td><td>Sample</td></tr><tr><td>1</td><td>Ladder</td></tr><tr><td>2</td><td>D1 - Part 1</td></tr><tr><td>3</td><td>D2 - Part 2-1</td></tr><tr><td>4</td><td>D3 - Part 2-2</td></tr></table><p><b>Notes:</b> New purple ladder and dye used<br /><b>Stop Time:</b> 8:00pm<br /><b>Next:</b> Try again, no band</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/7/2015<br />People in lab: Kent Gorday</b><br /><b>CT and miniprep of pSB4A5</b><br /><b>Start Time:</b> 9:00pm<br /><b>Purpose:</b> obtain pSB4A5 and clone plasmids<br /><b>Protocol:</b> Two minipreps were prepared, but the first was lost. 7/7 MP2 used 3mL of culture. Digests: D1 - 12.5 µL water, 2.5 tango, 8µL MP2, 1µL E, 1µL P<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>7/7 MP2</td><td>RFP pSB4A5</td><td>7/6 BC3</td><td>1</td></tr><tr><td>7/7 D1</td><td>psb4a5 cut e&p</td><td>7/7 MP2</td><td>1</td></tr><tr><td>7/7 CT1</td><td>hmp/pro/rbs amp</td><td>7/6 L1</td><td>1</td></tr><tr><td>7/7 CT2, 3, 4</td><td>Part1, 2-1, 2-2 on chlor</td><td>7/6 HiFi part 1, 2-1, 2-2</td><td>4</td></tr></table><p><b>Results:</b> MP2 - [DNA]: 371, 260/280: 1.76, 260/230: 1.36<br /><b>Stop Time:</b> 12:13am<br /><b>Next:</b> Run gel of 7/7 and 7/2 A1, inoculate BCs from CTs</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/8/2015<br />People in lab: Kent Gorday</b><br /><b>Inoculation of BCs</b><br /><b>Start Time:</b> 9:30am<br /><b>Purpose:</b> clone hmp/pro/rbs amp<br /><b>Protocol:</b> LM ed2<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>7/8 BC1, 2</td><td>hmp/pro/rbs amp</td><td>7/7 CT1</td><td>2</td></tr><tr><td>7/8 BC3, 4</td><td>hmp/pro/rbs amp</td><td>7/2 CT2</td><td>2</td></tr></table><p><b>Stop Time:</b> 9:50am<br /><b>Next:</b> Miniprep BCs, run gel of 7/7 D1 and 7/2 A1</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/9/2015<br />People in lab: Kent Gorday</b><br /><b>Minirpep, digest, and gel of pSB4A5, HiFi assemblies and hmp/pro/rbs</b><br /><b>Start Time:</b> 4:00pm<br /><b>Purpose:</b> obtain plasmids, verify backbone, parts, and assemblies<br /><b>Protocol:</b> Kitless miniprep procedure, LM ed. 2. Digests: D1 - 14.5µL MP1, 6µL water, 2.5 tango, 1 E, 1 P; D2 - 6µL MP3, 14.5 water, 2.5 tango, 1 E, 1 P. Ligation: 1µL buffer, 3.8 µL 7/9 D1, 4.2 6/30 D1, 1µL ligase<br /><b>Products:</b><br /></p><table><tr><td>Well</td><td>Sample</td></tr><tr><td>1</td><td>Hifi part 1 7/6 5µL</td></tr><tr><td>2</td><td>purple ladder, 10µL</td></tr><tr><td>3</td><td>7/2 A1, 10µL</td></tr><tr><td>4</td><td>7/7 D1, 10µL</td></tr><tr><td>5</td><td>7/9 D2, 10 µL</td></tr><tr><td>6</td><td>7/9 D1, 10µL</td></tr><tr><td>7</td><td>Hifi part 2-1 7/6 5µL</td></tr><tr><td>8</td><td>Hifi part 2-2 7/6 5µL</td></tr></table><p><b>Results:</b> MP1 - [DNA]: 172.9ng/µL, 260/280: 1.79, 260/230: 1.38; MP2 - 72, 1.81, 1.45; MP3 - 496.9, 1.82, 1.55.<br /><img class="gel" src="https://static.igem.org/mediawiki/2015/c/c1/Mstigem_jul9.jpeg" alt="gel electrophoresis" /><br /><b>Stop Time:</b> 11:55pm<br /><b>Next:</b> Analyze gel to determine if PCR product shoµLd be purified and ligated and transformed.</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/11/2015<br />People in lab: Kent Gorday</b><br /><b>tansformation efficiency and hmp/pro/rbs assembly</b><br /><b>Start Time:</b> 6:10pm<br /><b>Purpose:</b> Assess transformation efficiency of new electrocomp cells and assemble hmp/pro/rbs<br /><b>Protocol:</b> LM ed2<br /><b>Products:</b></p><table><tr><td>Sample Lable</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>7/11 ET1, 2</td><td>pro/rbs/hmp in chlor</td><td>7/9 L1</td><td>4</td></tr><tr><td>7/11 ET3</td><td>hmp/pro/rbs in amp</td><td>7/6 L1</td><td>2</td></tr><tr><td>7/11 ET4, 5</td><td>Trans eff. kit. 2015 10pg/µL</td><td>RFP in chlor</td><td>4</td></tr></table><p><b>Stop Time:</b> 7:30pm<br /><b>Next:</b> calculate efficiency, Miniprep , obtain GST plasmid, digest it and 7/2 A1</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/12/2015<br />People in lab: Kent Gorday</b><br /><b>Chemical transformation of hmp/pro/rbs</b><br /><b>Start Time:</b> 8:25pm<br /><b>Purpose:</b> to clone hmp/pro/rbs for later use<br /><b>Protocol:</b> LM ed2<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>7/12 CT1, 2</td><td>pro/rbs/hmp in chlor</td><td>7/9 L1</td><td>4</td></tr><tr><td>7/12 CT3</td><td>pro/hmp/rbs in amp</td><td>7/6 L1</td><td>2</td></tr></table><p><b>Notes:</b> 7/11 ET5 had a single red colony, all other empty<br /><b>Stop Time:</b> 11:40pm<br /><b>Next:</b> make new electrocomp and SOC, incoluate and miniprep growth</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/13/2015<br />People in lab: Kira Buckowing</b><br /><b>HiFi Assembly</b><br /><b>Start Time:</b> 6:45pM<br /><b>Purpose:</b> Correctly assemble plasmids for project work<br /><b>Protocol:</b> HiFi dna assembly protocol (same as last time except correct MM used)<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>HiFi part 1 7/13</td><td>Part 1</td><td>gblocks + pSB1C3</td></tr><tr><td>HiFi part 2-1 7/13</td><td>Part 2-1</td><td>gblocks + pSB1C3</td></tr><tr><td>HiFi part 2-2 7/13</td><td>Part 2-2</td><td>gblocks + pSB1C3</td></tr></table><p><b>Stop Time:</b> 7:15pm<br /><b>Next:</b> Trasformation to check</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/13/2015<br />People in lab: Kent Gorday</b><br /><b>CT of HiFis</b><br /><b>Start Time:</b> 8:00pm<br /><b>Purpose:</b> to clone and screen HiFi assemblies<br /><b>Protocol:</b> LM ed2<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>7/13 CT1</td><td>Part 1 on chlor+glucose</td><td>7/13 HiFi Part1</td><td>2</td></tr><tr><td>7/13 CT2</td><td>part 2-1 chlor on glucose</td><td>7/13 HiFi Part 2-1</td><td>2</td></tr><tr><td>7/13 CT3</td><td>part 2-2 chlor on glucose</td><td>7/13 HiFi Part 2-2</td><td>2</td></tr></table><p><b>Stop Time:</b> 11:20pm<br /><b>Next:</b> Inoculate BCs from CTs, miniprep</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/14/2015<br />People in lab: Kent Gorday</b><br /><b>Restriction digest and gel electrophoresis of HiFi assemblies</b><br /><b>Start Time:</b> 3:30pm<br /><b>Purpose:</b> Verify hiFi assembly products and transform<br /><b>Protocol:</b> LM ed2, Digests: D1 - 10µL HiFi part 1, 10.5 µL water, 2.5µL tango, 1 E, 1 P; D2 - 10µL part 2-1, 10.5 water, 2.5 tango, 1 E, 1 P; D3 - 10µL Part 2-2, 10.5 µL water, 2.5 tango, 1µL E, 1µL P<br /><b>Products:</b><br /></p><table><tr><td>Well</td><td>Sample</td></tr><tr><td>2</td><td>Purple, 2-log ladder</td></tr><tr><td>3</td><td>7/14 D1 10µL</td></tr><tr><td>4</td><td>7/14 D2 10µL</td></tr><tr><td>5</td><td>7/14 D3 10µL</td></tr><tr><td>6</td><td>7/6 L1 5µL</td></tr><tr><td>7</td><td>7/9 L1 5µL</td></tr></table><p><b>Results:</b><br /><img class="gel" src="https://static.igem.org/mediawiki/2015/c/cd/Mstigem_jul14.jpeg" alt="gel electrophoresis" /><br /><b>Stop Time:</b> 6:30pm<br /><b>Next:</b> Transform 7/13 HiFi part 1, 2-1, 2-2</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/14/2015<br />People in lab: Kira Buckowing</b><br /><b>CTs</b><br /><b>Start Time:</b> 5pm<br /><b>Purpose:</b> Transform the wanted plasmids into ecoli<br /><b>Protocol:</b> LM ed2<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>7/14 CT1</td><td> HiFi assemblies plasmids in e. coli</td><td>HiFi Part 1</td><td>2</td></tr><tr><td>7/14 CT2</td><td>HiFi assemblies plasmids in e. coli</td><td>HiFi Part 2-1</td><td>2</td></tr><tr><td>7/14 CT3</td><td>HiFi assemblies plasmidsin e. coli</td><td>HiFi Part 2-2</td><td>2</td></tr></table><p><b>Stop Time:</b> 7:10pm<br /><b>Next:</b> check for colonies</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/16/2015<br />People in lab: Kira Buckowing</b><br /><b>Inoculations</b><br /><b>Start Time:</b> 9:00pm<br /><b>Purpose:</b> To prepare liquid cultures for minipreps<br /><b>Protocol:</b> LM Ed2<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>BC1 7/16 KKB</td><td>Possibly Part1</td><td>7/14 CT1</td></tr><tr><td>BC2 7/16 KKB</td><td>Possibly Part1</td><td>7/14 CT1</td></tr></table><p><b>Stop Time:</b> 9:15 pm<br /><b>Next:</b> minipreps</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/17/2015<br />People in lab: Kent Gorday</b><br /><b>Miniprep, digest and gel of part 1 in psb1c3</b><br /><b>Start Time:</b> 3:10pm<br /><b>Purpose:</b> obtain and verify Part1 and pSB1C3<br /><b>Protocol:</b> LM ed2, three glycerol stocks were prepared, three kitless minipreps. Digest: D1 - 20.5µL MP1, 2.5µL 10x tango, 1µL E, 1µL P<br /><b>Products:</b><br /></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>7/17 MP1</td><td>Possible part 1 in chlor, [DNA]:109.37, 260/280:1.82, 260/230: 1.8</td><td>7/16 BC1</td></tr><tr><td>7/17 MP2</td><td>Possible Part 1 in chlor, [DNA]: 98.44, 260/280:1.79, 260/230:1.73</td><td>7/16 BC2</td></tr><tr><td>7/17 MP3</td><td>Part 1 in chlor, [DNA]: 67, 260/280:1.73, 260/230: 1.04</td><td>7/16 BC2</td></tr><tr><td>7/17 D1</td><td>Possible part 1 cut E&P</td><td>7/17 MP1</td></tr></table><p><b>Results:</b><br /><img class="gel" src="https://static.igem.org/mediawiki/2015/b/b6/Mstigem_jul17.jpeg" alt="gel electrophoresis" /><br /><b>Stop Time:</b> 9:00pm<br /><b>Next:</b> Inoculate new BCs and try minipreps again</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/20/2015<br />People in lab: Kent Gorday</b><br /><b>Transformation efficient of new electrocomp cells</b><br /><b>Start Time:</b> 3:00pm<br /><b>Purpose:</b> To test new electro comp cells and clone part 1<br /><b>Protocol:</b> Two BCs were made, two ETs were performed, LM ed2<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>7/20 BC1, 2</td><td>Possible part 1</td><td>7/16 BC1, 2</td><td>2</td></tr><tr><td>7/20 ET1, 2</td><td>RFP in chlor</td><td>Trans. eff. kit</td><td>4</td></tr></table><p><b>Stop Time:</b> 4:40pm<br /><b>Next:</b> calculate transformation efficiency and transform hifi assemblies</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/21/2015<br />People in lab: Kent Gorday</b><br /><b>Miniprep, digest, Transformation of HiFi assemblies</b><br /><b>Start Time:</b> 1:30pm<br /><b>Purpose:</b> transform HiFi assemblies into competent cells<br /><b>Protocol:</b> LM Ed2. Digests: D1 - 2.5µL tango, 1 E, 1 P, 20.5 µL 7/21 MP1<br /><b>Products:</b><br /></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>7/21 MP1, 2</td><td>Poss. part1</td><td>7/20 MP2</td><td>2</td></tr><tr><td>7/21 D1</td><td>Poss.part1 cut E&P</td><td>7/21 MP1</td><td>1</td></tr><tr><td>7/21 CT1</td><td>part1</td><td>Part 1</td><td>1</td></tr><tr><td>7/21 CT2</td><td>part2-1</td><td>Part2-1</td><td>1</td></tr><tr><td>7/21 CT3</td><td>part2-1</td><td>Part2-2</td><td>1</td></tr></table><p><b>Results:</b> MP1 [DNA]: 50, 260/280: 1.82, 260/230: 1.36; MP2 [DNA]: 17, 1.9, 1.8<br /><b>Stop Time:</b> 9:00pm<br /><b>Next:</b> continue transforming, pcr assembled parts to verify</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/22/2015<br />People in lab: Kent Gorday</b><br /><b>PCR of HiFi assemblies</b><br /><b>Start Time:</b> 2:01am<br /><b>Purpose:</b> verify hiFi assembly<br /><b>Protocol:</b> LM ed2, PCR: A1 - 10µL taq MM, 0.5 VR, 0.5 VF2, 9µL 7/13 hifi part1<br /><b>Exceptions:</b> PCR program: 94C 3min, 32 cycles of (94 26s, 55 40s, 68 3min), 68C 10min, 4C hold<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>7/22 A1</td><td>PCR of part 1</td><td>7/13 HiFi Part1</td></tr></table><p><b>Stop Time:</b> 2:41 am<br /><b>Next:</b> run gel of PCR product</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/22/2015<br />People in lab: Kent Gorday</b><br /><b>PCR and transformation of HiFi assemblies</b><br /><b>Start Time:</b> 2:30pm<br /><b>Purpose:</b> verify and transform assemblies<br /><b>Protocol:</b> LM ed2. PCR: A2 - 10µL MM, 0.5 VR, 0.5 VF, 9 HiFi part 2-1, A3 - 10µL Taq MM, 9µL 50pg/µL trans. eff. kit, 0.5 VR, 0.5 VF2<br /><b>Exceptions:</b> PCR program: 94C 5min, 32 cycles of (94 26s, 58 40s, 68 4min), 68 8min, 4C hold. CTs were performed with 25µL of commercial comp cells<br /><b>Products:</b><br /></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>7/22 A2</td><td>Part 2-1 pcr'd</td><td>HiFi part 2-1, 7/13</td></tr><tr><td>7/22 A3</td><td>positive control for pcr</td><td>Trans. eff. kit 50pg/µL</td></tr><tr><td>7/22 CT1</td><td>part1</td><td>7/13 HiFi part1</td></tr><tr><td>7/22 CT2</td><td>part2-1</td><td>7/13 HiFi part2-1</td></tr><tr><td>7/22 CT3</td><td>part2-2</td><td>7/13 HiFi part2-2</td></tr></table><p><b>Results:</b><br /><img class="gel" src="https://static.igem.org/mediawiki/2015/f/f3/Mstigem_jul22_1.jpeg" alt="gel electrophoresis" /><br /><img class="gel" src="https://static.igem.org/mediawiki/2015/8/84/Mstigem_jul22_2.jpeg" alt="gel electrophoresis" /><br /><b>Notes:</b> 7/22 CT4, 7/6 L1, hmp/pro/rbs in amp<br /><b>Stop Time:</b> 9:05pm<br /><b>Next:</b> Run gel of 7/22 A3</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/23/2015<br />People in lab: Kent Gorday</b><br /><b>Gel of part 2-2 amplified</b><br /><b>Start Time:</b> 6:20pm<br /><b>Purpose:</b> verify and amplify HiFis<br /><b>Protocol:</b> LM ed2<br /><b>Products:</b><br /></p><p><b>Results:</b><br /><img class="gel" src="https://static.igem.org/mediawiki/2015/d/db/Mstigem_jul23_1.jpeg" alt="gel electrophoresis" /><br /><img class="gel" src="https://static.igem.org/mediawiki/2015/6/6f/Mstigem_jul23_2.jpeg" alt="gel electrophoresis" /><br /><b>Stop Time:</b> 8:00pm<br /><b>Next:</b> miniprep, digest and ligate BCs into pSB1C3</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/24/2015<br />People in lab: Kent Gorday</b><br /><b>Miniprep, PCR, digest of Part 2-1</b><br /><b>Start Time:</b> 11:25am<br /><b>Purpose:</b> <br /><b>Protocol:</b> LM ed2, PCR: A1 - 10µL Taq MM, 1µL HiFi part 2-1, 0.25µL VR, 0.25µL 0.1x iGEM VF2, 8.5 water. Digest: D1 - 1µL E, 1µL P, 2.5µL tango, 20.5 µL MP2. Ligation: L1- 1µL buffer, 1µL ligase, 4.6µL ???, 3.4 µL ???<br /><b>Exceptions:</b> PCR program: 94C 5min, 32 cycles (94C 30s, 58C 30s, 68 5.5min), 68C 8min, 4C hold<br /><b>Products:</b><br /></p><table><tr><td>Well</td><td>Sample</td></tr><tr><td>2</td><td>purple ladder 5µL</td></tr><tr><td>4</td><td>7/24 D1 10µL</td></tr><tr><td>6</td><td>7/24 A1 10µL</td></tr></table><p><b>Results:</b> MP1 - [DNA]: 50, 260/280: 1.83, 260/230: 3; MP2 - [DNA]: 70, 260/280: 1.89, 260/230: 1.8<br /><b>Stop Time:</b> 6:20pm<br /><b>Next:</b> re-do hifi assemblies, transform 7/24 L1</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/25/2015<br />People in lab: Kent Gorday</b><br /><b>transformation of final pro/rbs/hmp</b><br /><b>Start Time:</b> 5:35pm<br /><b>Purpose:</b> clone final hmp/pro/rbs<br /><b>Protocol:</b> LM ed2<br /><b>Exceptions:</b> Commercial comp cells used<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>7/25 CT1</td><td>hmp/pro/rbs</td><td>7/24 L1</td><td>2</td></tr></table><p><b>Stop Time:</b> 8:30pm<br /><b>Next:</b> Inoculate liquid broth and miniprep</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/26/2015<br />People in lab: Kent Gorday</b><br /><b>Inoculation of liquid cultures of hmp/pro/rbs</b><br /><b>Start Time:</b> 3:10pm<br /><b>Purpose:</b> clone final hmp/pro/rbs<br /><b>Protocol:</b> LM ed2<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>7/26 BC1</td><td>hmp/pro/rbs in chlor</td><td>7/25 CT1</td><td>2</td></tr></table><p><b>Stop Time:</b> 3:25pm<br /><b>Next:</b> Miniprep, digst and gel to confirm</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/27/2015<br />People in lab: Kent Gorday</b><br /><b>Miniprep, digest of final part pro/rbs/hmp</b><br /><b>Start Time:</b> 7:30pm<br /><b>Purpose:</b> obtain final hmp part<br /><b>Protocol:</b> Kitless miniprep protocol, Digest: D1 - 20.5µL 7/27 MP1, 2.5 tnago buffer, 1µL E, 1µL P<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>7/27 MP1, 2</td><td>hmp/pro/rbs in chlor</td><td>7/26 BC1, 2</td><td>2</td></tr><tr><td>7/27 D1</td><td>hmp/pro/rbs in chlor</td><td>7/27 MP1</td><td>1</td></tr></table><p><b>Results:</b> MP1 - [DNA]: 100, 260/280: 1.71, 260/230: 1.3; MP2 - 100, 1.6, 1<br /><b>Stop Time:</b> 9:30pm<br /><b>Next:</b> run gel of 7/27 D1</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 7/28/2015<br />People in lab: Kent Gorday</b><br /><b>Gel of final hmp/pro/rbs</b><br /><b>Start Time:</b> 11:30 am<br /><b>Purpose:</b> verify identity of final hmp/pro/rbs<br /><b>Protocol:</b> LM ed2<br /><b>Products:</b><br /></p><p><b>Results:</b><br /><img class="gel" src="https://static.igem.org/mediawiki/2015/a/a8/Mstigem_jul28.png" alt="gel electrophoresis" /><br /><b>Stop Time:</b> 1:30pm<br /><b>Next:</b> Retry electrocomp cells (hmp part is correct)</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 8/18/2015<br />People in lab: Kira Buckowing</b><br /><b>PCR of Ocimene synthase</b><br /><b>Start Time:</b> 5:25pm<br /><b>Purpose:</b> Amplify gblock from IDT to digest/ligation<br /><b>Protocol:</b> LM ed2, digests: D1 - 12.5µL Taq MM, 5.5 water, 5µL ocimene synthase, 1µL VF2, 1µL VR<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>Ocimene PCR 8/18</td><td>Pcr'd ocimene synthase</td><td>Ocimene Synthase 6/30</td></tr></table><p><b>Stop Time:</b> <br /><b>Next:</b> Digestion and ligation for transformation</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 8/18/2015<br />People in lab: Kira Buckowing</b><br /><b>Digest and ligation of PCR'd ocimene sythase</b><br /><b>Start Time:</b> 8pm<br /><b>Purpose:</b> To transfer ocimene synthase gene to chlor BB<br /><b>Protocol:</b> LM ed2. Digest: D1 - 7µL DNA, 1µL P, 1µL E, 2.5 buffer, 13.5 water. Ligations: L1 - 2µL pSB1C3, 6µL D1, 1µL buffer, 1µL ligase; L2 - 4µL pSB1C3, 4µL D1, 1µL buffer, 1µL ligase<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>D1 8/18</td><td>digested amplified gblock</td><td>Ocimene Synthase PCR</td></tr><tr><td>L1 8/18</td><td>ligated ocimene + chlorBB</td><td>D1 + pSB1C3</td></tr><tr><td>L2 8/18</td><td> ligated ocimene + chlorBB</td><td>D1 + pSB1C3</td></tr></table><p><b>Stop Time:</b> 10pm<br /><b>Next:</b> Transform ligations</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 8/19/2015<br />People in lab: Kira Buckowing</b><br /><b>HiFi Control and transformations</b><br /><b>Start Time:</b> 2:45pm<br /><b>Purpose:</b> To ensure quality of HiFi kit, to trnasform ligations into e. coli<br /><b>Protocol:</b> HiFi as per HiFi Manual, Transformations as per LM ed2<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>HiFi Control 8/19</td><td>HiFi Control</td><td>HiFi Kit NEB</td><td>1</td></tr><tr><td>CT1 8/19</td><td>HiFi Control</td><td>HiFi Control 8/19</td><td>1</td></tr><tr><td>CT2, 3 8/19</td><td>Ocimene synthase</td><td>L1, L2, 8/18</td><td>4</td></tr></table><p><b>Notes:</b> Placed plates in G6A fridge 6:30pm 8/20 -LP<br /><b>Stop Time:</b> 5:20pm<br /><b>Next:</b> Check for success, make glycerol stocks or retry</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 8/24/2015<br />People in lab: Kira Buckowing</b><br /><b>BC Inoculations</b><br /><b>Start Time:</b> 5pm<br /><b>Purpose:</b> To grow cultures from plate colonies<br /><b>Protocol:</b> LM Ed2<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>BC1, 2 8/24 KKB</td><td>Possible ocimene synthase</td><td>CT2 8/19</td><td>2</td></tr><tr><td>BC3, 4 8/24 KKB</td><td>Possible ocimene synthase</td><td>CT3 8/19</td><td>2</td></tr></table><p><b>Stop Time:</b> 5:20pm<br /><b>Next:</b> Minipreps and nanodrops</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 8/25/2015<br />People in lab: Kira Buckowing</b><br /><b>Minipreps</b><br /><b>Start Time:</b> 6:30pm<br /><b>Purpose:</b> Purify plasmids<br /><b>Protocol:</b> Kitless miniprep procedure<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>MP1 8/25</td><td>Poss ocimene synthase. [DNA] 1167, 260/280: 1.73 260/230: 2.47</td><td>BC4 8/24</td></tr><tr><td>MP2 8/25</td><td>Poss ocimene synthase. 1411, 1.92, 2.47</td><td>BC2 8/24</td></tr><tr><td>MP3 8/25</td><td>Poss ocimene synthase. 929, 1.72, 2.42</td><td>BC3 8/24</td></tr></table><p><b>Stop Time:</b> 8:15pm<br /><b>Next:</b> Digest and check for correct</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 8/26/2015<br />People in lab: Kira Buckowing</b><br /><b>Digests and gel of ocimene synthase</b><br /><b>Start Time:</b> 3pm<br /><b>Purpose:</b> To verify ocimene synthase DNA<br /><b>Protocol:</b> LM ed2. D1 - 2.5 MP1, 2.5 buffer, 1 E, 1 P, 18 water; D2 - 2 MP2, 2.5 buffer, 1 µL E, 1 P, 18 water; D3 - 3µL MP3, 2.5 buffer, 1 E, 1 P, 18 µL water<br /><b>Products:</b><br /></p><table><tr><td>Well</td><td>Sample</td></tr><tr><td>1</td><td>ladder</td></tr><tr><td>2</td><td>D1</td></tr><tr><td>3</td><td>D2</td></tr><tr><td>4</td><td>D3</td></tr><tr><td>5</td><td>ladder</td></tr></table><p><b>Results:</b><br /><img class="gel" src="https://static.igem.org/mediawiki/2015/c/c8/Mstigem_aug26.jpeg" alt="gel electrophoresis" /><br /><b>Stop Time:</b> 6:00pm<br /><b>Next:</b> try again</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/1/2015<br />People in lab: Kira Buckowing</b><br /><b>Modified HiFi attempt</b><br /><b>Start Time:</b> 6:20pm<br /><b>Purpose:</b> Attempt to overcome high temp secondary structure in overhang regions of gblocks<br /><b>Protocol:</b> HiFi assembly protocol. Mixed 1-1 2.6µL, 1-2 3.1µL, 1-3 3.1µL, 1-4 3.6 µL, MM 13.7µL<br /><b>Exceptions:</b> Instead of 1hr at 50C, 25 min @ 50C, 10 min @ 60, 15 min @ 50C<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>Mod HiFi1 9/1 KKB</td><td>possibly assembled Part1</td><td>part 1 parts, BB</td></tr></table><p><b>Stop Time:</b> 7:30pm<br /><b>Next:</b> PCR to test for assembly</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/2/2015<br />People in lab: Levi Palmer</b><br /><b>Streaking pGEX4T-1 cells</b><br /><b>Start Time:</b> 4:00pm<br /><b>Purpose:</b> To grow cells containing the pGEX4T-1 plasmid for GST tagging<br /><b>Protocol:</b> 1. Wet sterile swab with LB, 2. Rub swab on bacterial culture, 3. Streak swab on new amp plate. Inoculate BC from swab.<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>8/2/15 pGEX4T-1 | 18|19</td><td>cells containing pGEX4T-1</td><td>pGEX 4T-1 18|19 box1 1/14</td></tr><tr><td>8/2/15 pGEX4T-1 B 18|19</td><td>cells containing pGEX4T-1</td><td>pGEX 4T-1 18|19 box1 1/14</td></tr></table><p><b>Stop Time:</b> 5pm<br /><b>Next:</b> Miniprep</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/2/2015<br />People in lab: Levi Palmer</b><br /><b>PCR and gel of hifi product and ocimene synthase</b><br /><b>Start Time:</b> 5pm<br /><b>Purpose:</b> Verify assembly of hifi, amplify ocimene synthase for standard assembly<br /><b>Protocol:</b> LM ed2. PCRs: A1 - 10.5 µL water, 12.5 Taq MM, 0.5 µL VR, 0.5 µL VF2, 1µL 5x10^9 diluted 9/1 hifi; A2 - 10.5 µL water, 12.5µL Taq MM, 0.5 µL VF2, 0.5 µL VR, 1µL 2x10^6 diluted MP2; A3 - 10.5µL water, 12.5µL Taq MM, 0.5 µL VR, 0.5 µL VF2, 1µL 0.5 pg/µL RFP DNA<br /><b>Exceptions:</b> PCR program: 94C 30s, 32 cycles of (95 20s, 57 20s, 68 4.25min), 68 5min, 4C hold<br /><b>Products:</b><br /></p><table><tr><td>Well</td><td>Sample</td></tr><tr><td>3</td><td>purple ladder</td></tr><tr><td>4</td><td>9/2 A1</td></tr><tr><td>5</td><td>9/2 A2</td></tr><tr><td>6</td><td>9/2 A3</td></tr></table><br /><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>9/2 A1</td><td>Part 1 top pathway amplified from HiFi assembly</td><td>9/1 HiFi 1</td></tr><tr><td>9/2 A2</td><td>ocimene synthase amplified from miniprep</td><td>8/25 MP2</td></tr><tr><td>9/2 A3</td><td>RFP pos control</td><td>0.5 pg/µL Trans Eff kit 2015</td></tr></table><p><b>Results:</b><br /><img class="gel" src="https://static.igem.org/mediawiki/2015/2/26/Mstigem_sep2.jpeg" alt="gel electrophoresis" /><br /><b>Stop Time:</b> 10:05pm<br /><b>Next:</b> Check ocimene pcr from 8/18</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/3/2015<br />People in lab: Kent Gorday</b><br /><b>Gel of ocimene pcr product</b><br /><b>Start Time:</b> 10:00am<br /><b>Purpose:</b> verify ocimene pcr<br /><b>Protocol:</b> LMed2<br /><b>Products:</b><br /></p><table><tr><td>Well</td><td>Sample</td></tr><tr><td>3</td><td>purple ladder</td></tr><tr><td>4</td><td>8/18 ocimene pcr</td></tr><tr><td>5</td><td>8/18 D1</td></tr></table><p><b>Results:</b><br /><img class="gel" src="https://static.igem.org/mediawiki/2015/5/5d/Mstigem_sep3.jpeg" alt="gel electrophoresis" /><br /><b>Stop Time:</b> 11:35am<br /><b>Next:</b> Determine why pcr failed</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/4/2015<br />People in lab: Levi Palmer</b><br /><b>Verification of 8/28 ocimene digests</b><br /><b>Start Time:</b> 10:30am<br /><b>Purpose:</b> To verify presence of DNA band from 8/26 digests of ocimene synthase<br /><b>Protocol:</b> LM ed2. Digests, D1 - 1000ng DNA, D2 - 2000ng, D3-3000ng, D4 - 2000ng, D5-1000ng, D6-2000ng, D7-3000ng, D8-2000ng. Rest of volume: 2.5µL 10x buffer, 1µL E, 1µL P, filled to 25µL with water<br /><b>Exceptions:</b> D4, D8 had no E or P<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>LP 9/4 D1, D2, D3</td><td>MP2 digested with E&P at various DNA concentrations</td><td>MP2 8/25 KKB</td><td>3</td></tr><tr><td>LP 9/4 D5, D6, D7</td><td>MP3 digested with E&P at various DNA concentrations</td><td>MP3 8/25 KKB</td><td>3</td></tr><tr><td>LP 9/4 D4, D8</td><td>Negative controls (No E or P)</td><td>MP2, MP3</td><td>1</td></tr></table><p><b>Notes:</b>Well: 1 - D1, 2 - D2, 3- D3, 4-D4, 5-Ladder, 6-empty, 7-D5, 8-D6, 9-D7, 10-D8 <br /><b>Stop Time:</b> 3pm<br /><b>Next:</b> Figure out why PCR failed</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/9/2015<br />People in lab: Kent Gorday</b><br /><b>PCR amplificaton of ocimene synthase, BCs of pGEX4T-1</b><br /><b>Start Time:</b> 2pm<br /><b>Purpose:</b> amplify ocimene synthase<br /><b>Protocol:</b> LM ed.2, PCRs: A1 - 10.5 water, 12.5 TaqMM, 0.5 VF2, 0.5 OcimeneR, 1µL 10^5 diluted ocimene gblock. Digest: D1 - 20 water, 2.5µL tango, 1 E, 1P 0.5 A1<br /><b>Exceptions:</b> PCR program: 95C 30s, 32 cycles (95C 20s, 57C 20s, 68C 2min), 68 5min, 4 hold<br /><b>Products:</b><br /></p><table><tr><td>Well</td><td>Sample</td></tr><tr><td>4</td><td>5µL ladder</td></tr><tr><td>5</td><td>10µL 9/9 A1</td></tr></table><br /><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>9/9 A1</td><td>amplified ocimene synthase</td><td>Part 3glock</td><td>1</td></tr><tr><td>9/9 D1</td><td>nothing</td><td>9/9 A1</td><td>1</td></tr><tr><td>9/9 BC1</td><td>pGEX4t-1 in amp</td><td>pGEX4t-1</td><td>3</td></tr><tr><td>9/9 A2</td><td>amplified ocimene synthase</td><td>part3 gblock</td><td>1</td></tr></table><p><b>Results:</b><br /><img class="gel" src="https://static.igem.org/mediawiki/2015/5/51/Mstigem_sep9_1.jpeg" alt="gel electrophoresis" /><br /><b>Notes:</b> Placed in red freezer box 8:00pm LP 9/9<br /><b>Stop Time:</b> 6pm<br /><b>Next:</b> run gel of A2</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/9/2015<br />People in lab: Kent Gorday</b><br /><b>Gel of ocimene synthase PCR</b><br /><b>Start Time:</b> 8pm<br /><b>Purpose:</b> verify PCR product<br /><b>Protocol:</b> LM Ed2<br /><b>Products:</b><br /></p><table><tr><td>Well</td><td>Sample</td></tr><tr><td>5</td><td>ladder</td></tr><tr><td>6</td><td>10µL 9/9 A2</td></tr></table><p><b>Results:</b><br /><img class="gel" src="https://static.igem.org/mediawiki/2015/b/b0/Mstigem_sep9_2.jpeg" alt="gel electrophoresis" /><br /><b>Stop Time:</b> 9:30pm<br /><b>Next:</b></p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/10/2015<br />People in lab: Levi Palmer</b><br /><b>PCR of ocimene synthase gblock</b><br /><b>Start Time:</b> 5:30pm<br /><b>Purpose:</b> to amplify gblock for digestion<br /><b>Protocol:</b> LM ed2, A1 - 10 water, 12.5 MM, 0.5 VF2, 0.5 ocimeneR, 1.5 gblock; A2 - 9 water, 12.5 MM, 0.5 VF2, 0.5 ocimeneR, 2.5 µL gblock<br /><b>Exceptions:</b> ran with igem/hmp pcr program<br /><b>Products:</b><br /></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>A1 9/10 LP</td><td>PCR amplified ocimene gblock</td><td>ocimene gblock</td></tr><tr><td>A2 9/10 LP</td><td>PCR amplified ocimene gblock</td><td>ocimene gblock</td></tr></table><p><b>Stop Time:</b> 6pm<br /><b>Next:</b> Gel to confirm</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/10/2015<br />People in lab: Kira Buckowing</b><br /><b>Gel of ocimene pcr</b><br /><b>Start Time:</b> 8:15pm<br /><b>Purpose:</b> confirm ocimene was amplified<br /><b>Protocol:</b> LM ed2<br /><b>Products:</b><br /></p><table><tr><td>Well</td><td>Sample</td></tr><tr><td>3</td><td>A1 LP 9/10</td></tr><tr><td>4</td><td>ladder</td></tr><tr><td>5</td><td>A2 LP 9/10</td></tr></table><p><b>Results:</b><br /><img class="gel" src="https://static.igem.org/mediawiki/2015/e/e9/Mstigem_sep10.jpeg" alt="gel electrophoresis" /><br /><b>Stop Time:</b> 9:10pm<br /><b>Next:</b> looks fine, new stuff again</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/11/2015<br />People in lab: Levi Palmer</b><br /><b>Final digest of ocimene block and bb</b><br /><b>Start Time:</b> 7:40pm<br /><b>Purpose:</b> to prepare ocimene for ligation<br /><b>Protocol:</b> lm ed2<br /><b>Exceptions:</b> 13µL final volume(buffer adjusted accordingly)<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>9/11 D1 LP</td><td>ocimene digested E&P</td><td>ocimene gblock</td></tr><tr><td>9/11 D2 LP</td><td>chlor bb digested E&P</td><td>PSB1C3 2015 linear</td></tr></table><p><b>Stop Time:</b> <br /><b>Next:</b> Ligation</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/11/2015<br />People in lab: Levi Palmer</b><br /><b>Final ligation of ocimene</b><br /><b>Start Time:</b> continued from prev<br /><b>Purpose:</b> to ligate ocimene into chlorbb<br /><b>Protocol:</b> LM ed2. <br /><b>Exceptions:</b> 1:1, 1:3, 1:5 molar ratios BB:Insert used<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>9/11 LP L1</td><td>1:1 chlorbb:ocimene</td><td>9/11 LP D1, D2</td></tr><tr><td>9/11 LP L2</td><td>1:3 chlorbb:ocimene</td><td>9/11 LP D1, D2</td></tr><tr><td>9/11 LP L3</td><td>1:5 chlorbb:ocimene</td><td>9/11 LP D1, D2</td></tr></table><p><b>Stop Time:</b> 10:20pm<br /><b>Next:</b> transform products and/or pcr</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/12/2015<br />People in lab: Levi Palmer</b><br /><b>PCR of ligations and gel</b><br /><b>Start Time:</b> 11am<br /><b>Purpose:</b> to check for complete oci+bb plasmids in ligations<br /><b>Protocol:</b> LM ed2<br /><b>Exceptions:</b> 0.3 µL of ligations used<br /><b>Products:</b><br /></p><table><tr><td>Well</td><td>Sample</td></tr><tr><td>2</td><td>A1</td></tr><tr><td>3</td><td>A2</td></tr><tr><td>4</td><td>ladder</td></tr><tr><td>5</td><td>A3</td></tr></table><br /><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>LP 9/12 A1</td><td>pcr of L1</td><td>9/11 L1</td></tr><tr><td>LP 9/12 A2</td><td>pcr of L2</td><td>9/11 L2</td></tr><tr><td>LP 9/12 A3</td><td>pcr of L3</td><td>9/11 L3</td></tr></table><p><b>Results:</b><br /><img class="gel" src="https://static.igem.org/mediawiki/2015/0/0c/Mstigem_sep12.png" alt="gel electrophoresis" /><br /><b>Stop Time:</b> <br /><b>Next:</b> gel extraction</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/12/2015<br />People in lab: Levi Palmer</b><br /><b>GE of ocimene synthase, PCR, gel</b><br /><b>Start Time:</b> cont from prev<br /><b>Purpose:</b> To purify pcrs from gel and amplify and confirm<br /><b>Protocol:</b> IBI GE procedure, LM ed2<br /><b>Exceptions:</b> A4 - 2µL GE used, A5 - 4, A6 - 8, A7 - 4. A7 neg control, no primer<br /><b>Products:</b><br /></p><table><tr><td>Well</td><td>Sample</td></tr><tr><td>2</td><td>A4</td></tr><tr><td>3</td><td>A5</td></tr><tr><td>4</td><td>ladder</td></tr><tr><td>5</td><td>A6</td></tr><tr><td>6</td><td>A7</td></tr></table><br /><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>LP GE1 9/12</td><td>combined, gel purified ocimene synthase dna</td><td>A1, A2, A3 9/12 LP</td><td>1</td></tr><tr><td>LP A4, A5, A6</td><td>amplified ocimene synthase</td><td>GE1 LP 9/12</td><td>3</td></tr><tr><td>LP 9/12 A7</td><td>neg control, no primer</td><td>GE1 LP 9/12</td><td>1</td></table><p><b>Results:</b> extensive smearing<br /><b>Notes:</b> GE has high salt conc.<br /><b>Stop Time:</b> <br /><b>Next:</b> Retry PCR of L1,2,3</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/12/2015<br />People in lab: Levi Palmer</b><br /><b>PCR of GE1 and L1 L2 L3, pcr purification of A9</b><br /><b>Start Time:</b> cont from prev<br /><b>Purpose:</b> To amplify ocimene synthase, IBI PCR purification protocol<br /><b>Protocol:</b> LM ed2<br /><b>Exceptions:</b> A8 - 1µL L1, A9 - 5/5µL L2/L3, A10 - 2µL L2, A11 - 1 L2<br /><b>Products:</b><br /></p><table><tr><td>Well</td><td>Sample</td></tr><tr><td>3</td><td>A11</td></tr><tr><td>4</td><td>ladder</td></tr><tr><td>5</td><td>A8</td></tr><tr><td>6</td><td>A9</td></tr><tr><td>7</td><td>A10</td></tr></table><br /><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>PCR Pure LP 9/12</td><td>ocimene synthase pcr amplified product</td><td>LP 9/12 A9</td></tr></table><p><b>Results:</b> smearing<br /><b>Stop Time:</b> <br /><b>Next:</b> PCR</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/12/2015<br />People in lab: Levi Palmer</b><br /><b>PCR of L1, L2, L3 and pcr pure 9/12 and dilution of primers, gel</b><br /><b>Start Time:</b> cont. from prev<br /><b>Purpose:</b> To amplify ocimene synthase<br /><b>Protocol:</b> VF2 - 0.16 mg+2.56 ml water, VR - 0.17 mg + 2.78ml water. LM ed2<br /><b>Exceptions:</b> A12 - 0.3 µL L2, A13 - 0.6 µL L2, A14 - 0.3µL L2, A15-0.3µL PCR Pure. A13 and A14 are double volume(MM adjusted accordingly)<br /><b>Products:</b><br /></p><table><tr><td>Well</td><td>Sample</td></tr><tr><td>1</td><td>A12</td></tr><tr><td>2</td><td>A13</td></tr><tr><td>3</td><td>A14</td></tr><tr><td>4</td><td>ladder</td></tr><tr><td>5</td><td>A15</td></tr></table><br /><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>LP 9/12 A12,13,14</td><td>amplified ocimene synthase</td><td>L2 LP 9/12</td><td>3</td></tr><tr><td>LP 9/12 A15</td><td>amplified ocimene synthase</td><td>PCR pure LP 9/12</td><td>1</td></tr><tr><td>VF2 LP 9/15</td><td>10uM VF2 primer</td><td>IDT VF2</td><td>2</td></tr><tr><td>VR LP 9/15</td><td>10uM VR primer</td><td>IDT VR</td><td>2</td></tr></table><p><b>Results:</b><br /><img class="gel" src="https://static.igem.org/mediawiki/2015/f/fa/Mstigem_sep13_1.png" alt="gel electrophoresis" /><br /><b>Notes:</b> primers in both red and clear freezer boxes<br /><b>Stop Time:</b> 1:30 am<br /><b>Next:</b> Final PCR attempt, CT of L1, L2, L3</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/12/2015<br />People in lab: Levi Palmer</b><br /><b>Final PCR of ocimene synthase</b><br /><b>Start Time:</b> cont. from prev<br /><b>Purpose:</b> Final PCR of ocimene synthase to eliminate smearing<br /><b>Protocol:</b> LM ED2<br /><b>Exceptions:</b> ALC - 0.2 µL L1 used<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td></tr><tr><td>ALC LP 9/12</td><td>amplified ocimene synthase</td><td>L1 9/12 LP</td></tr></table><br /><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>9/13 CT1,4,5</td><td>ocimene synthase cells?</td><td>L1 LP 9/11</td><td>6</td></tr><tr><td>CT2 9/13</td><td>ocimene synthase cells?</td><td>L2 LP 9/11</td><td>2</td></tr><tr><td>CT3 9/13</td><td>ocimene synthase cells?</td><td>L3 LP 9/11</td><td>2</td></tr><tr><td>BC1,2,3,4,5</td><td>3µL CT broth in LB+chlor</td><td>CT1,2,3,4,5</td><td>5</td></table><p><b>Stop Time:</b> 2:30am<br /><b>Next:</b> gel</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/13/2015<br />People in lab: Kira Buckowing</b><br /><b>CT of L1-3 9/12 LP</b><br /><b>Start Time:</b> 4:30pm<br /><b>Purpose:</b> To check for complete plasmids<br /><b>Protocol:</b> NEB chemical comp protocol<br /><b>Exceptions:</b> Used chemical comp cells<br /><b>Stop Time:</b> 7:20pm<br /><b>Next:</b> Hope for miracle</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/14/2015<br />People in lab: Kira Buckowing</b><br /><b>BCs of CTs</b><br /><b>Start Time:</b> 5pm<br /><b>Purpose:</b> Grow cultures for MPs<br /><b>Protocol:</b> LM ed2<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>BC 1, 2, 3, 4, 5</td><td>BC of ocimene synthase?</td><td>CT2</td><td>5</td></tr><tr><td>BC 6, 7, 8, 9, 10, 11</td><td>ocimene synthase?</td><td>CT3</td><td>5</td></tr><tr><td>BC 12-21</td><td>ocimene synthase?</td><td>CT5</td><td>10</td></tr></table><p><b>Stop Time:</b> 5:50pm<br /><b>Next:</b> miniprep anything that grows</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/15/2015<br />People in lab: Kira Buckowing</b><br /><b>Minipreps</b><br /><b>Start Time:</b> 1:30pm<br /><b>Purpose:</b> Purify a plasmid of ocimene synthase<br /><b>Protocol:</b> Kitless miniprep procedure<br /><b>Products:</b></p><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>MP1-15</td><td>is it ocimene synthase?</td><td>BC1-15</td><td>15</td></tr></table><p><b>Stop Time:</b> 7:30pm<br /><b>Next:</b> Digest with e&p, run gel</p>
 +
</div>
 +
<div id="mstigem">
 +
<p><b>Date: 9/15/2015<br />People in lab: Kira Buckowing</b><br /><b>Digest and gel</b><br /><b>Start Time:</b> 7:50pm<br /><b>Purpose:</b> to check for ocimene synthase (1750bp)<br /><b>Protocol:</b> LM ed2<br /><b>Products:</b></p><table><tr><td>Well</td><td>Sample</td></tr><tr><td>1</td><td>D1/D9</td></tr><tr><td>2</td><td>D2/D10</td></tr><tr><td>3</td><td>D3/D11</td></tr><tr><td>4</td><td>D4/D12</td></tr><tr><td>5</td><td>L/L</td></tr><tr><td>6</td><td>D5/D13</td></tr><tr><td>7</td><td>D6/D14</td></tr><tr><td>8</td><td>D7/D15</td></tr></table><br /><table><tr><td>Sample Label</td><td>Description</td><td>Source Label</td><td>Quantity</td></tr><tr><td>KKB D1-15 9/15</td><td>Ocimene synthase cut E&P?</td><td>MP1-15 9/15 KKB</td><td>15</td></tr></table><p><b>Results:</b><br /><img class="gel" src="https://static.igem.org/mediawiki/2015/6/6f/Mstigem_sep15_1.jpeg" alt="gel electrophoresis" /><br /><img class="gel" src="https://static.igem.org/mediawiki/2015/3/3b/Mstigem_sep15_2.jpeg" alt="gel electrophoresis" /><br /><b>Stop Time:</b> 9:40PM<br /><b>Next:</b> This is the end.</p>
 
</div>
 
</div>
−
<canvas id="echoes" onmousemove="mouse(event)"></canvas>
 
−
<p id="credit" onmousemove="mouse(event)">Missouri cave photo by Lynn Dieter<br />Northern Long-Eared bat photo by Scott Bergeson<br />White-Nose Syndrome spread map by Lindsey Heffernan<br />Little Brown bat photo by Jonathan Mays</p>
 
  
−
<script type="text/javascript">
 
−
// using setInterval for support in IE8 and 9, rather than requestAnimationFrame
 
−
// ugly nested ifs are a result of mediawiki parsing &&
 
−
"use strict";
 
−
var canvas = document.getElementById("echoes");
 
−
var ctx = canvas.getContext("2d");
 
−
var mousex;
 
−
var mousey;
 
−
var reflectx, reflecty;
 
−
setInterval(sendEcho, 12, canvas, ctx);
 
  
−
function mouse(event) {
 
−
  mousex = event.clientX;
 
−
  mousey = event.clientY + document.body.scrollTop;
 
−
}
 
  
−
function sendEcho(canvas, ctx) {
+
 
−
  if(typeof sendEcho.radius == 'undefined') {
+
 
−
    sendEcho.radius = Math.round(Math.sqrt((canvas.width / 2)*(canvas.width / 2) + (canvas.height / 2)*(canvas.height / 2)) / 284);
+
 
−
    sendEcho.reflecting = false;
+
 
−
    sendEcho.rradius = Math.round(Math.sqrt((canvas.width / 2)*(canvas.width / 2) + (canvas.height / 2)*(canvas.height / 2)) / 284);
+
 
−
  }
+
 
−
  canvas.width = document.documentElement.clientWidth;
+
 
−
  canvas.height = document.documentElement.offsetHeight;
+
 
−
  ctx.clearRect(0, 0, canvas.width, canvas.height);
+
 
−
  ctx.beginPath();
+
 
−
  ctx.strokeStyle = "white";
+
 
−
  ctx.globalAlpha = 0.16;
+
 
−
  ctx.lineWidth = 2;
+
 
−
  ctx.arc(canvas.width / 8, 2 * canvas.height / 3, sendEcho.radius, 0, 2 * Math.PI);
+
 
−
  ctx.stroke();
+
 
−
  if(sendEcho.radius > Math.sqrt((canvas.width / 2)*(canvas.width / 2) + (canvas.height / 2)*(canvas.height / 2)) / 30) {
+
 
−
    ctx.beginPath();
+
 
−
    ctx.strokeStyle = "white";
+
 
−
    ctx.globalAlpha = 0.16;
+
 
−
    ctx.lineWidth = 2;
+
 
−
    ctx.arc(canvas.width / 8, 2 * canvas.height / 3, Math.round(sendEcho.radius - Math.sqrt((canvas.width / 2)*(canvas.width / 2) + (canvas.height / 2)*(canvas.height / 2)) / 30), 0, 2 * Math.PI);
+
 
−
    ctx.stroke();
+
 
−
  }
+
 
−
  if(sendEcho.radius > Math.sqrt((canvas.width / 2)*(canvas.width / 2) + (canvas.height / 2)*(canvas.height / 2)) / 15) {
+
 
−
    ctx.beginPath();
+
 
−
    ctx.strokeStyle = "white";
+
 
−
    ctx.globalAlpha = 0.16;
+
 
−
    ctx.lineWidth = 2;
+
 
−
    ctx.arc(canvas.width / 8, 2 * canvas.height / 3, Math.round(sendEcho.radius - Math.sqrt((canvas.width / 2)*(canvas.width / 2) + (canvas.height / 2)*(canvas.height / 2)) / 15), 0, 2 * Math.PI);
+
 
−
    ctx.stroke();
+
 
−
  }
+
 
−
  if(Math.abs(sendEcho.radius - Math.sqrt((mousex - canvas.width / 8) * (mousex - canvas.width / 8) + (mousey - 2 *canvas.height / 3)*(mousey - 2 *canvas.height / 3))) < Math.round(Math.sqrt((canvas.width / 2)*(canvas.width / 2) + (canvas.height / 2)*(canvas.height / 2)) / 284)) {
+
 
−
    if(!sendEcho.reflecting) {
+
 
−
      sendEcho.reflecting = true;
+
 
−
      reflectx = mousex;
+
 
−
      reflecty = mousey;
+
 
−
      sendEcho.rradius = Math.round(Math.sqrt((canvas.width / 2)*(canvas.width / 2) + (canvas.height / 2)*(canvas.height / 2)) / 284);
+
 
−
    }
+
 
−
  }
+
 
−
  if(sendEcho.reflecting) {
+
<p id="credit" >Missouri cave photo by Lynn Dieter</p>
−
    ctx.beginPath();
+
−
    ctx.strokeStyle = "white";
+
−
    ctx.globalAlpha = 0.16;
+
−
    ctx.lineWidth = 2;
+
−
    ctx.arc(reflectx, reflecty, sendEcho.rradius, Math.atan2(reflecty - 2 * canvas.height / 3, reflectx - canvas.width / 8) - Math.PI / 12 + Math.PI, Math.atan2(reflecty - 2 * canvas.height / 3, reflectx - canvas.width / 8) + Math.PI / 12 + Math.PI);
+
−
    ctx.stroke();
+
−
    if(sendEcho.rradius > Math.sqrt((canvas.width / 2)*(canvas.width / 2) + (canvas.height / 2)*(canvas.height / 2)) / 30) {
+
−
      ctx.beginPath();
+
−
      ctx.strokeStyle = "white";
+
−
      ctx.globalAlpha = 0.16;
+
−
      ctx.lineWidth = 2;
+
−
      ctx.arc(reflectx, reflecty, Math.round(sendEcho.rradius - Math.sqrt((canvas.width / 2)*(canvas.width / 2) + (canvas.height / 2)*(canvas.height / 2)) / 30), Math.atan2(reflecty - 2 * canvas.height / 3, reflectx - canvas.width / 8) - Math.PI / 12 + Math.PI, Math.atan2(reflecty - 2 * canvas.height / 3, reflectx - canvas.width / 8) + Math.PI / 12 + Math.PI);
+
−
      ctx.stroke();
+
−
    }
+
−
    if(sendEcho.rradius > Math.sqrt((canvas.width / 2)*(canvas.width / 2) + (canvas.height / 2)*(canvas.height / 2)) / 15) {
+
−
      ctx.beginPath();
+
−
      ctx.strokeStyle = "white";
+
−
      ctx.globalAlpha = 0.16;
+
−
      ctx.lineWidth = 2;
+
−
      ctx.arc(reflectx, reflecty, Math.round(sendEcho.rradius - Math.sqrt((canvas.width / 2)*(canvas.width / 2) + (canvas.height / 2)*(canvas.height / 2)) / 15), Math.atan2(reflecty - 2 * canvas.height / 3, reflectx - canvas.width / 8) - Math.PI / 12 + Math.PI, Math.atan2(reflecty - 2 * canvas.height / 3, reflectx - canvas.width / 8) + Math.PI / 12 + Math.PI);
+
−
      ctx.stroke();
+
−
    }
+
−
    if(Math.round(Math.sqrt((canvas.width / 2)*(canvas.width / 2) + (canvas.height / 2)*(canvas.height / 2)) / 284) != 0) {
+
−
      sendEcho.rradius += Math.round(Math.sqrt((canvas.width / 2)*(canvas.width / 2) + (canvas.height / 2)*(canvas.height / 2)) / 284);
+
−
    }
+
−
    else {
+
−
      sendEcho.rradius++;
+
−
    }
+
−
    if(sendEcho.rradius > Math.sqrt(canvas.width*canvas.width+canvas.height*canvas.height) * 1.3) {
+
−
      sendEcho.reflecting = false;
+
−
    }
+
−
  }
+
−
  if(Math.round(sendEcho.radius - Math.sqrt((canvas.width / 2)*(canvas.width / 2) + (canvas.height / 2)*(canvas.height / 2)) / 15) > Math.sqrt((7 * canvas.width / 8)*(7 * canvas.width / 8) + (2 * canvas.height / 3)*(2 * canvas.height / 3))) {
+
−
    if(!sendEcho.reflecting) {
+
−
      sendEcho.radius = Math.round(Math.sqrt((canvas.width / 2)*(canvas.width / 2) + (canvas.height / 2)*(canvas.height / 2)) / 284);
+
−
    }
+
−
  }
+
−
  if(Math.round(Math.sqrt((canvas.width / 2)*(canvas.width / 2) + (canvas.height / 2)*(canvas.height / 2)) / 284) != 0) {
+
−
    sendEcho.radius += Math.round(Math.sqrt((canvas.width / 2)*(canvas.width / 2) + (canvas.height / 2)*(canvas.height / 2)) / 284);
+
−
  }
+
−
  if(sendEcho.rradius >= Math.sqrt((reflectx - canvas.width / 8)*(reflectx - canvas.width / 8)+(reflecty - 2 * canvas.height / 3)*(reflecty - 2 * canvas.height / 3))) {
+
−
    if(sendEcho.rradius <= Math.sqrt(canvas.width*canvas.width+canvas.height*canvas.height)) {
+
−
      if(canvas.width > 800) {
+
−
        ctx.beginPath();
+
−
        ctx.fillStyle = "#d7b351";
+
−
        ctx.globalAlpha = 0.6;
+
−
        ctx.arc(29 * canvas.width / 240, 43 * canvas.height / 63, 2, 0, 2*Math.PI);
+
−
        ctx.fill();
+
−
        ctx.beginPath();
+
−
        ctx.fillStyle = "#d7b351";
+
−
        ctx.globalAlpha = 0.6;
+
−
        ctx.arc(31 * canvas.width / 240, 43 * canvas.height / 63, 2, 0, 2*Math.PI);
+
−
        ctx.fill();
+
−
      }
+
−
      else {
+
−
        ctx.beginPath();
+
−
        ctx.fillStyle = "#d7b351";
+
−
        ctx.globalAlpha = 0.6;
+
−
        ctx.arc(28 * canvas.width / 240, 43 * canvas.height / 63, 1, 0, 2*Math.PI);
+
−
        ctx.fill();
+
−
        ctx.beginPath();
+
−
        ctx.fillStyle = "#d7b351";
+
−
        ctx.globalAlpha = 0.6;
+
−
        ctx.arc(31 * canvas.width / 240, 43 * canvas.height / 63, 1, 0, 2*Math.PI);
+
−
        ctx.fill();
+
−
      }
+
−
    }
+
−
  }
+
−
  else {
+
−
    sendEcho.radius++;
+
−
  }
+
−
}
+
−
</script>
+
 
</html>
 
</html>

Latest revision as of 05:09, 20 November 2015

Missouri cave photographed by Lynn Dieter

NOTEBOOK

Many of our standard procedures can be found in our Lab Training Manual.

Date: 5/22/2015
People in lab: Kent Gorday
Electrical Transformation of 2015 iGEM Plate 1 3O
Start Time: 3:00pm
Purpose: To transform and clone a promoter and rbs in standard iGEM backbone for later addition of last year's hmp part.
Protocol: 1.5µL of BBa_K608002, previously taken from iGEM Plate 1 3O, was electrically transformed into competent cell. An 1.8kV pulse was applied according to Lab Manual procedure. Plates were left to incubate overnight.
Products:

Sample LabelDescriptionSource Label
5/22 ET1 KG 20µLBBa_K608002 in E. coli on chloramphenicol, 20 µL plating volume2015 iGEM Plate 1 3O
5/22 ET1 KG 200µLBBa_K608002 in E. coli on chloramphenicol, 200 µL plating volume2015 iGEM Plate 1 3O

Stop Time: 4:10pm
Next: Inoculate liquid culture from 5/22 ET1 then miniprep product and glycerol stock of hmp.

Date: 5/25/2015
People in lab: Kira Buckowing
InocµLations of hmp frozen stock and colonies from ET1 5/22 200µL plate
Start Time: 3:00pm
Purpose: To have liquid culture grown up of hmp and the iGEM kit parts (Promoter + rbs)
Protocol: Inoculation of 2 broth cultures from each the hmp frozen stock and the ET1 5/22 200µL plate. Wire loop was used for 3 of the 4 total samples, pipette for the last hmp broth culture. Procedure taken from iGEM LTP Lab Manual edition 2
Products:

Sample LabelDescriptionSource LabelQuantity
BC1 KKB 5/25iGEM kit part, liquid cultureET1 5/222
BC3 KKB 5/25hmp stock liquid culturehmp glycerol stock2

Stop Time: 3:25pm
Next: Minipreps of all samples to isolate the desired plasmids.

Date: 5/26/2015
People in lab: Kent Gorday
Miniprep and digestion of hmp, pro/rbs, and amp BB
Start Time: 3:09pm
Purpose: To extract and digest BBa_K608002, hmp, and linearized pSB1A3
Protocol: Solution miniprep procedure attached to front of lab training manual was used on 3mL of liquid culture. Minipreps 1-4 were analyzed by nanodrop and showed no DNA due to a mistake in miniprep procedure. Identical minipreps were performed using the remaining 2mL of culture. Nanodrop then showed DNA presence, so the following digests were prepared: D1- 20.5 µL pSB1A3, 2.5 µL 10x Tango, 1 µL EcoRI, 1 µL PstI; D2 - 14.5 µL water, 2.5 µL 10x Tango, 6 µL MP5, 1 µL EcoRI, 1 µL SpeI; D3 - 18.5 µL water, 2.5 µL 10x Tango, 2 µL MP7, 1 µL XbaI, 1 µL PstI
Products:

Sample LabelDescriptionSource LabelQuantity
KG 5/26 MP1, MP2, MP5, MP6pro/rbs BBa_K680025/25 BC1, BC24
KG 5/26 MP3, MP4, MP7, MP8hmp5/25 BC1, BC24
KG 5/26 D1E&P amp BBLinear pSB1A3 20151
KG 5/26 D2E&S pro/rbs5/26 MP51
KG 5/26 D3X&P hmp5/26 MP71

Results: MP1 - [DNA]: -4.9, 260/280: 1.65, 260/230: 0.06; MP2 - [DNA]: -2.2, 260/280: 1.28, 260/230: 0.04; MP3 - [DNA]: 13.8, 260/280: 1.38, 260/230: 0.35; MP4 - [DNA]: -4.8, 260/280: 1.65, 260/230: 0.08; MP5 - [DNA]: 161.8, 260/280: 1.91, 260/230: 2.51; MP6 - [DNA]: 150, 260/280: 1.87, 260/230: 2.28; MP7 - [DNA]: 508.5, 260/280: 1.85, 260/230: 1.84; MP8 - [DNA]: 462.9, 260/280: 1.82, 260/230: 1.55
Stop Time: 7:50pm
Next: Ligate 5/26 D1, D2, D3

Date: 5/27/2015
People in lab: Kira Buckowing
Ligation of hmp/pro/rbs/amp BB
Start Time: 3:15pm
Purpose: To combine the wanted digest parts from 5/26 into one plasmid (3A assembly)
Protocol: Ligation protocol from LM ed2 with the following adjustments: 1µL each of the hmp section and pro/rbs section used for L1 & 2µL each used for L2 with 4µL of the amp backbone
Products:

Sample LabelDescriptionSource Label
L1 KKB 5/271µL D2, 1 µL D3, 6µL D1; ligated plasmid5/26 KG D1, D2, D3
L2 KKB 5/272µL D2, 1 µL D3, 6µL D1; ligated plasmid5/26 KG D1, D2, D3

Stop Time: 4:25PM
Next: Test plasmid with gel electrophoresis or go straight to transformation to see if the plasmid ligated as planned

Date: 5/28/2015
People in lab: Kent Gorday
Electrical transformation of pro/rbs+hmp+amp
Start Time: 3:00pm
Purpose: To transform functioning hmp plasmid into competent cells for cloning
Protocol: 2µL of the ligations from 5/27 were transformed into electrocompetent cells using a 1.8kV pµLse and LM procedure. Plates were left to incubate overnight.
Products:

Sample LabelDescriptionSource LabelQuantity
5/28 ET1hmp+pro/rbs on amp plate, no arc5/27 L12
5/28 ET2hmp+pro/rbs on amp plate, arced5/27 L12
5/28 ET3hmp+pro/rbs on amp plate, no arc5/27 L22
5/28 ET3hmp+pro/rbs on amp plate, arced5/27 L22

Stop Time: 4:15pm
Next: Inoculate liquid cultures from transformations then miniprep

Date: 6/7/2015
People in lab: Kent Gorday
Inoculation of liquid cultures amp+hmp+pro/rbs
Start Time: 4:25pm
Purpose: To clone hmp+pro/rbs/amp plasmid for later miniprep
Protocol: According to inoculation protocol, 5µL of ampicillin was added to each 5µL broth culture, then left to incubate overnight
Products:

Sample LabelDescriptionSource LabelQuantity
6/7 KG BC11x amp, amp/pro/rbs5/28 ET1 20µL4

Stop Time: 4:55pm
Next: Miniprep cultures and digest

Date: 6/8/2015
People in lab: Kent Gorday
Observation
Start Time: 2:20pm
Purpose: Observe broth cultures
Notes: Cultures from 6/7 show only slight growth and were left to incubate again overnight. Preparation of miniprep solutions was completed and solutions put in refrigerator,
Stop Time: 3:35pm
Next: Miniprep cultures and digest

Date: 6/9/2015
People in lab: Kent Gorday
Inoculation of liquid cultures
Start Time: 11:25am
Purpose: To clone hmp/pro/rbs+amp for later miniprep
Protocol: 5µL of amp was added to each 5mL LB tube, then a sterile pipette tip used to inoculate the cultures near a flame. Cultures were left in shaking incubator.
Products:

Sample LabelDescriptionSource LabelQuantity
KG 6/9 BC11x amp, hmp+pro+rbsKG 5/28 ET2 20µL2

Stop Time: 11:40am
Next: Miniprep cultures and digest

Date: 6/10/2015
People in lab: Kent Gorday
Miniprep, digest, ligation of hmp/pro/rbs
Start Time: 12:50pm
Purpose: To produce a completed pro/rbs/hmp part for submission
Protocol: Three solution minipreps were performed using 1.5mL of 6/7 BC3 and BC4, and 3mL of 6/9BC1. The amounts of solutions 1,2,3 used per miniprep were 150, 200, and 150µL, respectively. DNA was resuspended in 30µL of TE buffer. Digests: D1 - 10.5µL milliQ water, 2.5µL Tango, 1µL EcoRI, 1 µL PstI, 10µL 6/10MP1; D2 - 3.5µL MilliQ, 2.5µL Tango, 1 µL EcoRI, 1 µL PstI, 17µL 6/10 MP3; D3 - 2.5µL Tango, 1µL EcoRI, 1µL PstI, 20.5 µL pSB1C3 2015. Ligations: L1 - 6µL D3, 2µL D1,1µL 10x T4 buffer, 1µL T4 ligase; L2 - 6µL D3, 2µL D2, 1µL 10x buffer, 1µL T4 ligase; L3 - 4 µL D3, 4µL D1, 1µL 10x buffer, 1µL T4 ligase; L4 - 4µL D3, 4µL D2, 1µL 10x buffer, 1µL T4 ligase
Products:

Sample LabelDescriptionSource LabelQuantity
KG 6/10 MP1,2hmp+pro/rbs+amp6/7 BC3, 42
KG 6/10 MP3hmp+pro/rbs+amp6/9 BC11
6/10 KG D1, 2pro/rbs+hmp cut E&P6/10 KG MP1, 32
6/10 D3 KGchlorBB cut E&PpSB1C3 20151
6/10 KG L1, 2, 3, 4completed hmp+pro/rbs part for igem6/10 D3&D1, D2&D3, D3&D1, D3&D24

Results: MP1 - [DNA]: 100, 260/280: 1.82, 260/230: 1.84; MP2 - [DNA]: 98.5, 260/280: 1.81, 260/230: 1.93; MP3 - [DNA]: 60.7, 260/280: 1.84; 260/230: 2.35
Stop Time: 5:00pm
Next: Transform ligations to clone and miniprep

Date: 6/11/2015
People in lab: Kira Buckowing, Kent Gorday
Electrical transformation of hmp/rbs part and 2J from kit
Start Time: 4:40pm
Purpose: To grow colonies with the wanted plasmids
Protocol: 2J DNA resuspended in 10µL milliQ, ETs per lab manual
Products:

Sample LabelDescriptionSource LabelQuantity
2J KKB 6/11/15Part BBa_J04450 in pSB4A5 backbone, rfp in ampiGEM DNA Plate 4 well 2J1
6F KKB 6/11/15Part BBa_J04450 in pSB3K3 bb, rfp in kaniGEM DNA plate 4 well 6F1
KG 6/11 ET1, ET2, ET3, ET4ET of L1, L1, L2, L2, L3 L3KG 6/10 L1, L1, L2, L2, L3, L38
ET5 KKB 6/112J Kit part ETET5 of 2J2
ET6 KKB 6/116F Kit part ETET6 of 6F2

Notes: ET6 arced. No growth -KKB 6/13
Stop Time: 6:20pm
Next: Check for growth

Date: 6/16/2015
People in lab: Kent Gorday
New electrical transformations of hmp/pro/rbs and 2J, 6F kit parts
Start Time: 4:56 pm
Purpose: To transform parts fro cloning
Protocol: LM ed. 2, D1 through D4 - 17.5µL milliQ, 2.5 Tango, 1µL PstI, and 4µL of 6/10 L1 through L4, respectively.
Products:

Sample LabelDescriptionSource LabelQuantity
6/16 D1, 2, 3, 4hmp/pro/rbs digested with PKG 6/10 L1, L2, L3, L44
6/16 ET1, 2hmp/pro/rbs on chlor, plated in duplicates 20 and 200µLKG 6/10 L1, L24
6/16 ET3J04450 on amp, plated in duplicates 20/200µLKKB 6/11 2J2
6/16 ET4J04450 on kan, plated in duplicates 20/200µLKKB 6/11 6F2

Stop Time: 6:35pm
Next: If hmp plates grow, prepare part for iGEM submission. Otherwise run gel electrophoresis of digests to diagnose.

Date: 6/17/2015
People in lab: Kent Gorday
Gel electrophoresis of hmp/pro/rbs digested with PstI
Start Time: 4:50pm
Purpose: To troubleshoot hmp/pro/rbs using gel electrophoresis
Protocol: LM ed2
Products:

WellSample
35µL low range ladder
46/16 D1 15µL
56/16 D2 15µL
66/16 D3 15µL
76/16 D4 15µL

Results:
gel electrophoresis
Notes: Calculated fragment lengths for all ligations: 2300bp, 4000bp
Stop Time: 6:48pm
Next: Run gel of MP 5/26 and digests from 6/10

Date: 6/18/2015
People in lab: Kent Gorday
Gel electrophoresis of 6/10 digests and 5/26 MP
Start Time: 7:20pm
Purpose: To continue troubleshooting hmp/pro/rbs using gel electrophoresis
Protocol: LM ed2, Digests: D1 - 11.5 MilliQ, 2.5µL Tango, 10µL 5/26 P1, 1µL PstI
Products:

Well Sample
2 5µL low range ladder
3 6/16 D1, 10µL
4 6/18 D1, 15µL
5 6/10 D1, 10µL
6 6/10 D3 15µL

Stop Time: 11:00pm
Next: Repeat transformation of 5/27 L1 & 2 with new ampicillin plates

Date: 6/19/2015
People in lab: Kent Gorday
Gel purification and ligation of hmp/pro/rbs
Start Time: 6:00pm
Purpose: To assemble pro/rbs/hmp using gel purification
Protocol: LM ed2, Digest D1 - 20.5 µL 2015 pSB1C3, 2.5 µL Tango, 1µL EcoRI, 1µL PstI, Ligation: L1 - 4µL 6/19 GE1, 2µL GE2, 2µL GE3, 1µL buffer, 1µL T4 ligase
Products:

WellSample
16/19 D1, 15µL
25/26 D2, 15µL
35/26 D3, 15µL
4Low range ladder 5µL

Results: Nanodrop showed no DNA in gel purifications.
Stop Time: 10:45pm
Next: Repeat transformation of 5/27 L1 & 2 on new ampicillin plates

Date: 6/20/2015
People in lab: Kent Gorday
Electrical transformations of hmp+pro/rbs on amp/chlor
Start Time: 2:52pm
Purpose: to clone hmp+pro/rbs plasmids
Protocol: 1µL of ligations transformed into ecomp cells
Products:

Sample LabelDescriptionSource LabelQuantity
6/20 ET1hmp/pro/rbs on amp5/27 L12
6/20 ET2hmp/pro/rbs on amp5/27 L22
6/20 ET3hmp/pro/rbs on chlor6/20 L12

Stop Time: 4:00pm
Next: Inoculate liquid cultures and miniprep

Date: 6/22/2015
People in lab: Kent Gorday
Inoculation of liquid cultures of hmp/pro/rbs/amp
Start Time: 9:52am
Purpose: To clone plasmid for later miniprep
Protocol: 5µL amp added to tubes, inoculated with sterile pipette tips
Products:

Sample LabelDescriptionSource LabelQuantity
6/22 BC16/20 ET22

Stop Time: 10:10am
Next: Miniprep and possibly find different amp plates

Date: 6/22/2015
People in lab: Kira Buckowing
ET of 2J and hmp/amp
Start Time: 4:35 pm
Purpose: To transform wanted plasmids into ecoli for later use
Protocol: LM ed2
Exceptions: 2µL each of hmp used
Products:

Sample LabelDescriptionSource LabelQuantity
ET1 6/22 KKB2J kit parts, RFP2J KKB2
ET2 6/22 KKBhmp/pro/rbs in amphmp L1 5/272
ET3 6/22 KKBhmp/pro/rbs in amphmp L2 5/272

Notes: ET3 popped, ET2 was spilled
Stop Time: 6:35pm
Next: Inoculation of broth cultures

Date: 6/23/2015
People in lab: Kira Buckowing
Inoculation of pro/rbs/hmp, 2J, 6F kit parts
Start Time: 4:45pm
Purpose: To obtain liquid cultures for minipreps
Protocol: LM ed2
Products:

Sample LabelDescriptionSouce LabelQuantity
ET3 BC1 KKBET3 6/221
ET BC2 KKB1ET2 6/221
ET1C BC3, 4 KKBcolony from lid of dish 2J2
BC5, 6 KKBcolony of 6F2

Stop Time: 5pm
Next: miniprep

Date: 6/24/2015
People in lab: Kent Gorday
Start Time: 7:55pm
Purpose: To assemble pro/rbs/hmp/chlor and ensure identity of product
Protocol: Kitless minipreps procedure. Digests: D1 - 18.5 milliq, 2.5 tango, 2 6/24 MP1, 1 EcoRI, 1 PstI; D2 - 10.5 milliq, 2.5 tango, 10 6/24 MP3, 1µL EcoRI, 1µL PstI; D3 - 10.5milliq, 2.5 tango, 10 pSB1C3 2014, 1µL EcoRI, 1µL PstI. Ligation: L1 - 5µL D3, 3µL D2, 1µL buffer, 1µL ligase
Exceptions: 150µL of soln 1, 200 µL of soln 2, 175µL of sln 3, 550µL isopropyl alcohol.
Products:

Sample LabelDescriptionSouce LabelQuantity
6/24 MP12J plate 4, lid colony 6/23 BC4ET1C1
6/24 MP2, 3hmp/pro/rbs amp 6/22 BC1, 22
6/24 ET1, 2hmp/pro/rbs/amp5/27 L1,24
6/24 D12J digested E&P 6/24 MP11
6/24 D2hmp/pro/rbs digested E&P6/24 MP31

Results: MP1 - [DNA]: 524.4ng/µL, 260/280: 1.85, 260/230: 2.32; MP3 - [DNA]: 96/73, 260/280: 1.82, 260/230: 1.75
Notes: 6/24 D3,pSB1C3 2014, linear B digested E&P; 6/24 L1, 6/24 D2&D3, hmp/pro/rbs chlor
Stop Time: 12:01am
Next: Run gel of digests and transform ligations if lengths are correct.

Date: 6/25/2015
People in lab: Kent Gorday
Gel elecetrophoresis of MPs
Start Time: 4:30pm
Purpose: to ensure identity of miniprep products
Protocol: LM ed2
Products:

WellSample
35µL low range ladder
410µL 6/24 D1
510µL 6/24 D2

Stop Time: 5:40pm
Next: religate hmp/pro/rbs amp

Date: 6/27/2015
People in lab: Kent Gorday
New ligation of hmp/pro/rbs+amp
Start Time: 8:55pm
Purpose: To porform new ligations of 5/26 D1, 2, and 3 since gel electrophoresis showed failed of 5/27 ligations
Protocol: the following ligations were prepared and left to react overnight, L1 - 5/26 D1 7µL, 0.8µL D3, 0.2µL D2, 1µL 10x buffer, 1µL T4 ligase
Products:

Sample LabelDescriptionSource Label
6/27 L1hmp/pro/rbs + amp5/26 D1, D2, D3

Stop Time: 9:35 pm
Next: Transform ligation, culture, and miniprep

Date: 6/28/2015
People in lab: Kent Gorday
Electrical transformation of kit parts and hmp/pro/rbs/amp
Start Time: 6:05pm
Purpose: to clone plasmids for later inoculation and miniprep
Protocol: 10µL MilliQ water was used to resuspend 2014 plate 4 6F. Four electrical transformations were prepared according to the lab manual.
Products:

Sample LabelDescriptionSource LabelQuantity
6/28 ET1, 2hmp/pro/rbs/amp2µL 6/27 L12
6/28 ET32J on amp1µL 2015-4-2J1
6/28 ET46F on kan1µL 2014-4-6F1

Stop Time: 7:30pm
Next: Inoculate liquid cultures

Date: 6/30/2015
People in lab: Kira Buckowing
gBlock DNA resuspension and restriction digest
Start Time: 4:40pm
Purpose: To get gBlock DNA into a usable form to prep the pSB1C3 backbone for project work.
Protocol: Digest as per Lab Manual and resuspensions as per IDT instructions: 1. Centrifuge tube for a few seconds 2. add TE up to 10 ng/µL 3. Vortex briefly 4. Incubate @ 50C for 20 min 5. Vortex and centrifuge again briefly 6. [Added step] transfer total to pcr tube
Products:

Sample LabelDescriptionSource LabelQuantity
pSB1C3 Digested 6/30pSB1C3 cut with E&PpSB1C3 kit DNA1
6/30/15 KKB 1-1, 1-2, 1-3, 1-410ng/µL DNA Part 1 1of4, 2of4, 3of4, 4of44
6/30/15 KKB, 2.1-1,2.1-2,2.1-3,2.1-410ng/µL DNA Part 2-1 1of4, 2of4, 3of4, 4of44
6/30/15 KKB, 2.2-1,2.2-2,2.2-3,2.2-410ng/µL DNAPart 2-2 1of4, 2of4, 3of4, 4of44
Part3 6/30/15 KKB10ng/µL DNAPart 3 ocimene synthase1

Notes: 100µL added to 2.2-1 instead of the calculated 50µL so the concentration is halfed from the expected. 2014 linearized pSB1C3 used.
Stop Time: 7:00pm
Next: Assembly of the gBlock DNA fragments

Date: 6/30/2015
People in lab: Kent Gorday
Electrical transformation of Kit parts and hmp/pro/rbs+amp
Start Time: 8:30pm
Purpose: to clone plasmids for later inoculation and miniprep
Protocol: LM ed2
Products:

KG 6/30 ET3
Sample LabelDescriptionSource LabelQuantity
KG 6/30 ET1hmp/pro/rbs on amp6/27 L12
KG 6/30 ET2pSB4A5 on amp 2015 plate4 2J2
pSB3K3 on kan2014 plate4 6F12

Stop Time: 9:40pm
Next: inoculate broth cultures and miniprep

Date: 7/2/2015
People in lab: Kent Gorday
Chemical transformations ond PCR of hmp for gst tagging
Start Time: 8:27pm
Purpose: clone plasmids and prepare hmp for gst tagging
Protocol: LM ed2, PCR - A1: 12.5 µL Taq MM, 11.3µL MilliQ, 0.2µL 5/26 MP7, 0.5 µL F hmp, 0.5 µL R hmp, Ligation: L1 - 1µL 10x buffer, 5.1 µL 5/26 D1, 2.4 µL 5/26 D2, 0.5 µL 5/26 D3, 1µL ligase
Exceptions: PCR Program: 94C - 2mins, 32 cycles of (94C - 26s, 55C- 40s, 68C - 3min), 68C - 7min, 4C - hold
Products:

Sample LabelDescriptionSource LabelQuantity
7/2 BC12014 6F in kan6/30 ET32
7/2 BC3hmp in pSB1C3hmp glycerol stock1
7/2 A1hmp prepaged for gst tagging5/26 MP71
7/2 CT1hmp/pro/rbs/amp6/27 L11
2015 plate 4 well 2J2015 2J plate411

Notes: 7/2 L1, 5/26 D1. D2, D3, hmp/pro/rbs/amp
Stop Time: 1:11am
Next: Run a gel of A1, 7/2 L1, 6/27 L1, miniprep BCs incolµLate new BCs from Cts

Date: 7/3/2015
People in lab: Kent Gorday
Miniprep hmp and pSB3K3
Start Time: 3:30pm
Purpose: Obtain plasmids from culture
Protocol: Three minipreps were performed using the kitless miniprep protocol
Products:

Sample LabelDescriptionSource Label
7/3 MP1pSB3K3 in TE7/2 BC1
7/3 MP2pSB3K3 in TE7/2 BC2
7/3 MP3hmp in pSB1C37/2 BC3

Results: MP2 - [DNA]: 320.1 ng/µL, 260/280: 1.89, 260/230: 2.38; MP3 - 216.4, 1.75, 1.8
Stop Time: 5:00pm
Next: ET of hmp/pro/rbs

Date: 7/3/2015
People in lab: Kent Gorday
Electrical transformation of hmp/pro/rbs/amp
Start Time: 5:10pm
Purpose: assemble hmp in pSB1C3
Protocol: LM Ed2
Products:

Sample LabelDescriptionSource LabelQuantity
7/3 ET1hmp/pro/rbs amp7/2 L12

Stop Time: 6:22pm
Next: If no growth, run a new digestion of pSB1A3 and repeat 7/2 L1 using new BB. Inoculate BC and MPs from 7/2 CT2, then digest pSB3K3 and pSB4A5 to check by gel

Date: 7/4/2015
People in lab: Kent Gorday
Inoculation of liquid cultures for pSB4A5
Start Time: 10:14 am
Purpose: clone pSB4A5 for later miniprep
Protocol: Two broth cultures were inoculated using a wire loop
Products:

Sample LabelDescriptionSource LabelQuantity
7/4 BC1RFP in pSB4A57/2 CT22

Stop Time: 10:22am
Next: Miniprep broth cultures

Date: 7/5/2015
People in lab: Kent Gorday
Miniprep and digestion of pSB4A5, pSB3K3, pSB1A3
Start Time: 7:35pm
Purpose: prepare pSB4A5 and pSB3K3 for gel and use
Protocol: Kitless miniprep protocol and LM ed2, Digests: D1 - 1µL E, 1µL P, 2.5µL tango, 11.5 water, 9µL 7/3 MP2; D2 - 1 E, 1 P, 2.5 buffer, 20.5 7/5 MP2; D3 - 1 E, 1 P, 2.5 buffer, 20.4 pSB1A3
Products:

Sample LabelDescriptionSource LabelQuantity
7/5 MP1RFP in pSB4A57/4 BC11
7/5 MP2RFP in pSB4A57/4 BC21
7/5 D1, D2RFP in pSB3K3 cut E&P7/3 MP22
7/5 D3pSB1A3 cut E&P2015 Linear pSB1A31

Results: MP1 - [DNA]: 69.37, 260/280: 1.87, 260/230: 1.92; MP2 - 138.52, 1.71, 1.46
Stop Time: 10:30pm
Next: Run gel of digests, repeat 7/2 L1 using 7/5 D3

Date: 7/6/2015
People in lab: Kent Gorday
Ligation of pro/rbs/hmp amp
Start Time: 9:51am
Purpose: assemble hmp/pro/rbs part
Protocol: L1 - 1µL ligase, 1µL buffer, 1.4 µL D3, 5µL D2, 1.6µL D3
Products:

Sample LabelDescriptionSource Label
7/6 L1hmp/pro/rbs/amp7/5 D3, 5/26 D2, 5/26 D3

Stop Time: 10:10am
Next: Run gel of 7/5 digests, transform ligation

Date: 7/6/2015
People in lab: Kira Buckowing
HiFi assembly Part 1
Start Time: 10:15am
Purpose: To assemble part 1
Protocol: Following mixed in PCR tube and heated to 50C for 1 hr, HiFi - HiFi Part 1: 1.3µL pSB1C3, 1-1 2.6µL, 1-2 3.1µL, 1-3 3.1µL, 1-4 3.6µL, HiFi MM 13.2
Products:

Sample LabelDescriptionSource Label
HiFi part 1 7/6 KKBAssembled Part 1 for projectpSB1C3 6/30, 1-1, 1-2, 1-3, 1-4 6/30

Notes: Slightly less than an hour incubation
Stop Time: 11:25pm
Next: Tramsform and gel/digest to check product

Date: 7/6/2015
People in lab: Kent Gorday
Gel of backbone digests
Start Time: 11:28am
Purpose: To check identity of digests
Protocol: LM ed2
Products:

WellSample
3Ladder 5µL
47/5 D1 10µL
57/5 D2 10µL
67/5 D3 10µL

Stop Time: 12:55pm
Next: Transform 7/6 L1

Date: 7/6/2015
People in lab: Kira Buckowing
HiFi Assembly Part 2-1
Start Time: 1pm
Purpose: To assemble the Part 2-1
Protocol: The following were mixed in a PCR tube and incubated @ 50C for an hour: pSB1C3 1.3µL, 2.1-1 4.6µL, 2.1-2 2.8µL, 2.1-3 2.8µL, 2.1-4 3.1µL, HiFi MM 14.6 µL
Products:

Sample LabelDescriptionSource Label
HiFi Part 2.1 7/6 KKBPart 2-1 for projectpSB1C3 digested 6/30, 2.1-1, 2.1-2, 2.1-3, 2.1-4 6/30

Stop Time: 2:20pm
Next: Transformation into e. coli to check products

Date: 7/6/2015
People in lab: Kira Buckowing
Assembly of part 2-2
Start Time: 4:30pm
Purpose: To assemble the 2-2 part
Protocol: The following were mixed in a PCR tube and incubated at 50C for an hour. HiFi assembly: Part 2.2 - pSB1C3 1.3µL, 2.2-1 2.8µL, 2.2-2 2.8µL, 2.2-3 2.8µL, 2.2-4 3.1µL, HiFi MM 12.8µL
Products:

Sample LabelDescriptionSource Label
HiFi Part 2.2 7/6 KKB Part 2-2 for project work pSB1C3 6/30 2.2-1, 2, 3,4 6/30

Stop Time: 5:50pm
Next: Transformation and gel to check for product correctness

Date: 7/6/2015
People in lab: Kent Gorday
CT of HiFi assemblies and hmp/pro/rbs
Start Time: 7:10pm
Purpose: Transform HiFi assemblies to clone and verify plasmids and assembled hmp/pro/rbs part
Protocol: LM ed2
Products:

Sample LabelDescriptionSource LabelQuantity
7/6 BC1, 2, 3FP pSB4A57/2 CT23
7/6 ET1hmp/pro/rbs7/6 L12
7/6 ET2part 1 on chlorHiFi part 1 7/62
7/6 ET3part 2-1 on chlorHiFi part 2-1 7/62
7/6 ET4Part 2-2 on chlorHiFi part 2-2 7/62

Stop Time: 10:00 pm
Next: pSB4A5 and inoculte BC from transformations

Date: 7/7/2015
People in lab: Kira Buckowing
Digest and gel of HiFi assemblies
Start Time: 5:15pm
Purpose: To check validity of products
Protocol: LM ed 2
Products:

WellSample
1Ladder
2D1 - Part 1
3D2 - Part 2-1
4D3 - Part 2-2

Notes: New purple ladder and dye used
Stop Time: 8:00pm
Next: Try again, no band

Date: 7/7/2015
People in lab: Kent Gorday
CT and miniprep of pSB4A5
Start Time: 9:00pm
Purpose: obtain pSB4A5 and clone plasmids
Protocol: Two minipreps were prepared, but the first was lost. 7/7 MP2 used 3mL of culture. Digests: D1 - 12.5 µL water, 2.5 tango, 8µL MP2, 1µL E, 1µL P
Products:

Sample LabelDescriptionSource LabelQuantity
7/7 MP2RFP pSB4A57/6 BC31
7/7 D1psb4a5 cut e&p7/7 MP21
7/7 CT1hmp/pro/rbs amp7/6 L11
7/7 CT2, 3, 4Part1, 2-1, 2-2 on chlor7/6 HiFi part 1, 2-1, 2-24

Results: MP2 - [DNA]: 371, 260/280: 1.76, 260/230: 1.36
Stop Time: 12:13am
Next: Run gel of 7/7 and 7/2 A1, inoculate BCs from CTs

Date: 7/8/2015
People in lab: Kent Gorday
Inoculation of BCs
Start Time: 9:30am
Purpose: clone hmp/pro/rbs amp
Protocol: LM ed2
Products:

Sample LabelDescriptionSource LabelQuantity
7/8 BC1, 2hmp/pro/rbs amp7/7 CT12
7/8 BC3, 4hmp/pro/rbs amp7/2 CT22

Stop Time: 9:50am
Next: Miniprep BCs, run gel of 7/7 D1 and 7/2 A1

Date: 7/9/2015
People in lab: Kent Gorday
Minirpep, digest, and gel of pSB4A5, HiFi assemblies and hmp/pro/rbs
Start Time: 4:00pm
Purpose: obtain plasmids, verify backbone, parts, and assemblies
Protocol: Kitless miniprep procedure, LM ed. 2. Digests: D1 - 14.5µL MP1, 6µL water, 2.5 tango, 1 E, 1 P; D2 - 6µL MP3, 14.5 water, 2.5 tango, 1 E, 1 P. Ligation: 1µL buffer, 3.8 µL 7/9 D1, 4.2 6/30 D1, 1µL ligase
Products:

WellSample
1Hifi part 1 7/6 5µL
2purple ladder, 10µL
37/2 A1, 10µL
47/7 D1, 10µL
57/9 D2, 10 µL
67/9 D1, 10µL
7Hifi part 2-1 7/6 5µL
8Hifi part 2-2 7/6 5µL

Results: MP1 - [DNA]: 172.9ng/µL, 260/280: 1.79, 260/230: 1.38; MP2 - 72, 1.81, 1.45; MP3 - 496.9, 1.82, 1.55.
gel electrophoresis
Stop Time: 11:55pm
Next: Analyze gel to determine if PCR product shoµLd be purified and ligated and transformed.

Date: 7/11/2015
People in lab: Kent Gorday
tansformation efficiency and hmp/pro/rbs assembly
Start Time: 6:10pm
Purpose: Assess transformation efficiency of new electrocomp cells and assemble hmp/pro/rbs
Protocol: LM ed2
Products:

Sample LableDescriptionSource LabelQuantity
7/11 ET1, 2pro/rbs/hmp in chlor7/9 L14
7/11 ET3hmp/pro/rbs in amp7/6 L12
7/11 ET4, 5Trans eff. kit. 2015 10pg/µLRFP in chlor4

Stop Time: 7:30pm
Next: calculate efficiency, Miniprep , obtain GST plasmid, digest it and 7/2 A1

Date: 7/12/2015
People in lab: Kent Gorday
Chemical transformation of hmp/pro/rbs
Start Time: 8:25pm
Purpose: to clone hmp/pro/rbs for later use
Protocol: LM ed2
Products:

Sample LabelDescriptionSource LabelQuantity
7/12 CT1, 2pro/rbs/hmp in chlor7/9 L14
7/12 CT3pro/hmp/rbs in amp7/6 L12

Notes: 7/11 ET5 had a single red colony, all other empty
Stop Time: 11:40pm
Next: make new electrocomp and SOC, incoluate and miniprep growth

Date: 7/13/2015
People in lab: Kira Buckowing
HiFi Assembly
Start Time: 6:45pM
Purpose: Correctly assemble plasmids for project work
Protocol: HiFi dna assembly protocol (same as last time except correct MM used)
Products:

Sample LabelDescriptionSource Label
HiFi part 1 7/13Part 1gblocks + pSB1C3
HiFi part 2-1 7/13Part 2-1gblocks + pSB1C3
HiFi part 2-2 7/13Part 2-2gblocks + pSB1C3

Stop Time: 7:15pm
Next: Trasformation to check

Date: 7/13/2015
People in lab: Kent Gorday
CT of HiFis
Start Time: 8:00pm
Purpose: to clone and screen HiFi assemblies
Protocol: LM ed2
Products:

Sample LabelDescriptionSource LabelQuantity
7/13 CT1Part 1 on chlor+glucose7/13 HiFi Part12
7/13 CT2part 2-1 chlor on glucose7/13 HiFi Part 2-12
7/13 CT3part 2-2 chlor on glucose7/13 HiFi Part 2-22

Stop Time: 11:20pm
Next: Inoculate BCs from CTs, miniprep

Date: 7/14/2015
People in lab: Kent Gorday
Restriction digest and gel electrophoresis of HiFi assemblies
Start Time: 3:30pm
Purpose: Verify hiFi assembly products and transform
Protocol: LM ed2, Digests: D1 - 10µL HiFi part 1, 10.5 µL water, 2.5µL tango, 1 E, 1 P; D2 - 10µL part 2-1, 10.5 water, 2.5 tango, 1 E, 1 P; D3 - 10µL Part 2-2, 10.5 µL water, 2.5 tango, 1µL E, 1µL P
Products:

WellSample
2Purple, 2-log ladder
37/14 D1 10µL
47/14 D2 10µL
57/14 D3 10µL
67/6 L1 5µL
77/9 L1 5µL

Results:
gel electrophoresis
Stop Time: 6:30pm
Next: Transform 7/13 HiFi part 1, 2-1, 2-2

Date: 7/14/2015
People in lab: Kira Buckowing
CTs
Start Time: 5pm
Purpose: Transform the wanted plasmids into ecoli
Protocol: LM ed2
Products:

Sample LabelDescriptionSource LabelQuantity
7/14 CT1 HiFi assemblies plasmids in e. coliHiFi Part 12
7/14 CT2HiFi assemblies plasmids in e. coliHiFi Part 2-12
7/14 CT3HiFi assemblies plasmidsin e. coliHiFi Part 2-22

Stop Time: 7:10pm
Next: check for colonies

Date: 7/16/2015
People in lab: Kira Buckowing
Inoculations
Start Time: 9:00pm
Purpose: To prepare liquid cultures for minipreps
Protocol: LM Ed2
Products:

Sample LabelDescriptionSource Label
BC1 7/16 KKBPossibly Part17/14 CT1
BC2 7/16 KKBPossibly Part17/14 CT1

Stop Time: 9:15 pm
Next: minipreps

Date: 7/17/2015
People in lab: Kent Gorday
Miniprep, digest and gel of part 1 in psb1c3
Start Time: 3:10pm
Purpose: obtain and verify Part1 and pSB1C3
Protocol: LM ed2, three glycerol stocks were prepared, three kitless minipreps. Digest: D1 - 20.5µL MP1, 2.5µL 10x tango, 1µL E, 1µL P
Products:

Sample LabelDescriptionSource Label
7/17 MP1Possible part 1 in chlor, [DNA]:109.37, 260/280:1.82, 260/230: 1.87/16 BC1
7/17 MP2Possible Part 1 in chlor, [DNA]: 98.44, 260/280:1.79, 260/230:1.737/16 BC2
7/17 MP3Part 1 in chlor, [DNA]: 67, 260/280:1.73, 260/230: 1.047/16 BC2
7/17 D1Possible part 1 cut E&P7/17 MP1

Results:
gel electrophoresis
Stop Time: 9:00pm
Next: Inoculate new BCs and try minipreps again

Date: 7/20/2015
People in lab: Kent Gorday
Transformation efficient of new electrocomp cells
Start Time: 3:00pm
Purpose: To test new electro comp cells and clone part 1
Protocol: Two BCs were made, two ETs were performed, LM ed2
Products:

Sample LabelDescriptionSource LabelQuantity
7/20 BC1, 2Possible part 17/16 BC1, 22
7/20 ET1, 2RFP in chlorTrans. eff. kit4

Stop Time: 4:40pm
Next: calculate transformation efficiency and transform hifi assemblies

Date: 7/21/2015
People in lab: Kent Gorday
Miniprep, digest, Transformation of HiFi assemblies
Start Time: 1:30pm
Purpose: transform HiFi assemblies into competent cells
Protocol: LM Ed2. Digests: D1 - 2.5µL tango, 1 E, 1 P, 20.5 µL 7/21 MP1
Products:

Sample LabelDescriptionSource LabelQuantity
7/21 MP1, 2Poss. part17/20 MP22
7/21 D1Poss.part1 cut E&P7/21 MP11
7/21 CT1part1Part 11
7/21 CT2part2-1Part2-11
7/21 CT3part2-1Part2-21

Results: MP1 [DNA]: 50, 260/280: 1.82, 260/230: 1.36; MP2 [DNA]: 17, 1.9, 1.8
Stop Time: 9:00pm
Next: continue transforming, pcr assembled parts to verify

Date: 7/22/2015
People in lab: Kent Gorday
PCR of HiFi assemblies
Start Time: 2:01am
Purpose: verify hiFi assembly
Protocol: LM ed2, PCR: A1 - 10µL taq MM, 0.5 VR, 0.5 VF2, 9µL 7/13 hifi part1
Exceptions: PCR program: 94C 3min, 32 cycles of (94 26s, 55 40s, 68 3min), 68C 10min, 4C hold
Products:

Sample LabelDescriptionSource Label
7/22 A1PCR of part 17/13 HiFi Part1

Stop Time: 2:41 am
Next: run gel of PCR product

Date: 7/22/2015
People in lab: Kent Gorday
PCR and transformation of HiFi assemblies
Start Time: 2:30pm
Purpose: verify and transform assemblies
Protocol: LM ed2. PCR: A2 - 10µL MM, 0.5 VR, 0.5 VF, 9 HiFi part 2-1, A3 - 10µL Taq MM, 9µL 50pg/µL trans. eff. kit, 0.5 VR, 0.5 VF2
Exceptions: PCR program: 94C 5min, 32 cycles of (94 26s, 58 40s, 68 4min), 68 8min, 4C hold. CTs were performed with 25µL of commercial comp cells
Products:

Sample LabelDescriptionSource Label
7/22 A2Part 2-1 pcr'dHiFi part 2-1, 7/13
7/22 A3positive control for pcrTrans. eff. kit 50pg/µL
7/22 CT1part17/13 HiFi part1
7/22 CT2part2-17/13 HiFi part2-1
7/22 CT3part2-27/13 HiFi part2-2

Results:
gel electrophoresis
gel electrophoresis
Notes: 7/22 CT4, 7/6 L1, hmp/pro/rbs in amp
Stop Time: 9:05pm
Next: Run gel of 7/22 A3

Date: 7/23/2015
People in lab: Kent Gorday
Gel of part 2-2 amplified
Start Time: 6:20pm
Purpose: verify and amplify HiFis
Protocol: LM ed2
Products:

Results:
gel electrophoresis
gel electrophoresis
Stop Time: 8:00pm
Next: miniprep, digest and ligate BCs into pSB1C3

Date: 7/24/2015
People in lab: Kent Gorday
Miniprep, PCR, digest of Part 2-1
Start Time: 11:25am
Purpose:
Protocol: LM ed2, PCR: A1 - 10µL Taq MM, 1µL HiFi part 2-1, 0.25µL VR, 0.25µL 0.1x iGEM VF2, 8.5 water. Digest: D1 - 1µL E, 1µL P, 2.5µL tango, 20.5 µL MP2. Ligation: L1- 1µL buffer, 1µL ligase, 4.6µL ???, 3.4 µL ???
Exceptions: PCR program: 94C 5min, 32 cycles (94C 30s, 58C 30s, 68 5.5min), 68C 8min, 4C hold
Products:

WellSample
2purple ladder 5µL
47/24 D1 10µL
67/24 A1 10µL

Results: MP1 - [DNA]: 50, 260/280: 1.83, 260/230: 3; MP2 - [DNA]: 70, 260/280: 1.89, 260/230: 1.8
Stop Time: 6:20pm
Next: re-do hifi assemblies, transform 7/24 L1

Date: 7/25/2015
People in lab: Kent Gorday
transformation of final pro/rbs/hmp
Start Time: 5:35pm
Purpose: clone final hmp/pro/rbs
Protocol: LM ed2
Exceptions: Commercial comp cells used
Products:

Sample LabelDescriptionSource LabelQuantity
7/25 CT1hmp/pro/rbs7/24 L12

Stop Time: 8:30pm
Next: Inoculate liquid broth and miniprep

Date: 7/26/2015
People in lab: Kent Gorday
Inoculation of liquid cultures of hmp/pro/rbs
Start Time: 3:10pm
Purpose: clone final hmp/pro/rbs
Protocol: LM ed2
Products:

Sample LabelDescriptionSource LabelQuantity
7/26 BC1hmp/pro/rbs in chlor7/25 CT12

Stop Time: 3:25pm
Next: Miniprep, digst and gel to confirm

Date: 7/27/2015
People in lab: Kent Gorday
Miniprep, digest of final part pro/rbs/hmp
Start Time: 7:30pm
Purpose: obtain final hmp part
Protocol: Kitless miniprep protocol, Digest: D1 - 20.5µL 7/27 MP1, 2.5 tnago buffer, 1µL E, 1µL P
Products:

Sample LabelDescriptionSource LabelQuantity
7/27 MP1, 2hmp/pro/rbs in chlor7/26 BC1, 22
7/27 D1hmp/pro/rbs in chlor7/27 MP11

Results: MP1 - [DNA]: 100, 260/280: 1.71, 260/230: 1.3; MP2 - 100, 1.6, 1
Stop Time: 9:30pm
Next: run gel of 7/27 D1

Date: 7/28/2015
People in lab: Kent Gorday
Gel of final hmp/pro/rbs
Start Time: 11:30 am
Purpose: verify identity of final hmp/pro/rbs
Protocol: LM ed2
Products:

Results:
gel electrophoresis
Stop Time: 1:30pm
Next: Retry electrocomp cells (hmp part is correct)

Date: 8/18/2015
People in lab: Kira Buckowing
PCR of Ocimene synthase
Start Time: 5:25pm
Purpose: Amplify gblock from IDT to digest/ligation
Protocol: LM ed2, digests: D1 - 12.5µL Taq MM, 5.5 water, 5µL ocimene synthase, 1µL VF2, 1µL VR
Products:

Sample LabelDescriptionSource Label
Ocimene PCR 8/18Pcr'd ocimene synthaseOcimene Synthase 6/30

Stop Time:
Next: Digestion and ligation for transformation

Date: 8/18/2015
People in lab: Kira Buckowing
Digest and ligation of PCR'd ocimene sythase
Start Time: 8pm
Purpose: To transfer ocimene synthase gene to chlor BB
Protocol: LM ed2. Digest: D1 - 7µL DNA, 1µL P, 1µL E, 2.5 buffer, 13.5 water. Ligations: L1 - 2µL pSB1C3, 6µL D1, 1µL buffer, 1µL ligase; L2 - 4µL pSB1C3, 4µL D1, 1µL buffer, 1µL ligase
Products:

Sample LabelDescriptionSource Label
D1 8/18digested amplified gblockOcimene Synthase PCR
L1 8/18ligated ocimene + chlorBBD1 + pSB1C3
L2 8/18 ligated ocimene + chlorBBD1 + pSB1C3

Stop Time: 10pm
Next: Transform ligations

Date: 8/19/2015
People in lab: Kira Buckowing
HiFi Control and transformations
Start Time: 2:45pm
Purpose: To ensure quality of HiFi kit, to trnasform ligations into e. coli
Protocol: HiFi as per HiFi Manual, Transformations as per LM ed2
Products:

Sample LabelDescriptionSource LabelQuantity
HiFi Control 8/19HiFi ControlHiFi Kit NEB1
CT1 8/19HiFi ControlHiFi Control 8/191
CT2, 3 8/19Ocimene synthaseL1, L2, 8/184

Notes: Placed plates in G6A fridge 6:30pm 8/20 -LP
Stop Time: 5:20pm
Next: Check for success, make glycerol stocks or retry

Date: 8/24/2015
People in lab: Kira Buckowing
BC Inoculations
Start Time: 5pm
Purpose: To grow cultures from plate colonies
Protocol: LM Ed2
Products:

Sample LabelDescriptionSource LabelQuantity
BC1, 2 8/24 KKBPossible ocimene synthaseCT2 8/192
BC3, 4 8/24 KKBPossible ocimene synthaseCT3 8/192

Stop Time: 5:20pm
Next: Minipreps and nanodrops

Date: 8/25/2015
People in lab: Kira Buckowing
Minipreps
Start Time: 6:30pm
Purpose: Purify plasmids
Protocol: Kitless miniprep procedure
Products:

Sample LabelDescriptionSource Label
MP1 8/25Poss ocimene synthase. [DNA] 1167, 260/280: 1.73 260/230: 2.47BC4 8/24
MP2 8/25Poss ocimene synthase. 1411, 1.92, 2.47BC2 8/24
MP3 8/25Poss ocimene synthase. 929, 1.72, 2.42BC3 8/24

Stop Time: 8:15pm
Next: Digest and check for correct

Date: 8/26/2015
People in lab: Kira Buckowing
Digests and gel of ocimene synthase
Start Time: 3pm
Purpose: To verify ocimene synthase DNA
Protocol: LM ed2. D1 - 2.5 MP1, 2.5 buffer, 1 E, 1 P, 18 water; D2 - 2 MP2, 2.5 buffer, 1 µL E, 1 P, 18 water; D3 - 3µL MP3, 2.5 buffer, 1 E, 1 P, 18 µL water
Products:

WellSample
1ladder
2D1
3D2
4D3
5ladder

Results:
gel electrophoresis
Stop Time: 6:00pm
Next: try again

Date: 9/1/2015
People in lab: Kira Buckowing
Modified HiFi attempt
Start Time: 6:20pm
Purpose: Attempt to overcome high temp secondary structure in overhang regions of gblocks
Protocol: HiFi assembly protocol. Mixed 1-1 2.6µL, 1-2 3.1µL, 1-3 3.1µL, 1-4 3.6 µL, MM 13.7µL
Exceptions: Instead of 1hr at 50C, 25 min @ 50C, 10 min @ 60, 15 min @ 50C
Products:

Sample LabelDescriptionSource Label
Mod HiFi1 9/1 KKBpossibly assembled Part1part 1 parts, BB

Stop Time: 7:30pm
Next: PCR to test for assembly

Date: 9/2/2015
People in lab: Levi Palmer
Streaking pGEX4T-1 cells
Start Time: 4:00pm
Purpose: To grow cells containing the pGEX4T-1 plasmid for GST tagging
Protocol: 1. Wet sterile swab with LB, 2. Rub swab on bacterial culture, 3. Streak swab on new amp plate. Inoculate BC from swab.
Products:

Sample LabelDescriptionSource Label
8/2/15 pGEX4T-1 | 18|19cells containing pGEX4T-1pGEX 4T-1 18|19 box1 1/14
8/2/15 pGEX4T-1 B 18|19cells containing pGEX4T-1pGEX 4T-1 18|19 box1 1/14

Stop Time: 5pm
Next: Miniprep

Date: 9/2/2015
People in lab: Levi Palmer
PCR and gel of hifi product and ocimene synthase
Start Time: 5pm
Purpose: Verify assembly of hifi, amplify ocimene synthase for standard assembly
Protocol: LM ed2. PCRs: A1 - 10.5 µL water, 12.5 Taq MM, 0.5 µL VR, 0.5 µL VF2, 1µL 5x10^9 diluted 9/1 hifi; A2 - 10.5 µL water, 12.5µL Taq MM, 0.5 µL VF2, 0.5 µL VR, 1µL 2x10^6 diluted MP2; A3 - 10.5µL water, 12.5µL Taq MM, 0.5 µL VR, 0.5 µL VF2, 1µL 0.5 pg/µL RFP DNA
Exceptions: PCR program: 94C 30s, 32 cycles of (95 20s, 57 20s, 68 4.25min), 68 5min, 4C hold
Products:

WellSample
3purple ladder
49/2 A1
59/2 A2
69/2 A3

Sample LabelDescriptionSource Label
9/2 A1Part 1 top pathway amplified from HiFi assembly9/1 HiFi 1
9/2 A2ocimene synthase amplified from miniprep8/25 MP2
9/2 A3RFP pos control0.5 pg/µL Trans Eff kit 2015

Results:
gel electrophoresis
Stop Time: 10:05pm
Next: Check ocimene pcr from 8/18

Date: 9/3/2015
People in lab: Kent Gorday
Gel of ocimene pcr product
Start Time: 10:00am
Purpose: verify ocimene pcr
Protocol: LMed2
Products:

WellSample
3purple ladder
48/18 ocimene pcr
58/18 D1

Results:
gel electrophoresis
Stop Time: 11:35am
Next: Determine why pcr failed

Date: 9/4/2015
People in lab: Levi Palmer
Verification of 8/28 ocimene digests
Start Time: 10:30am
Purpose: To verify presence of DNA band from 8/26 digests of ocimene synthase
Protocol: LM ed2. Digests, D1 - 1000ng DNA, D2 - 2000ng, D3-3000ng, D4 - 2000ng, D5-1000ng, D6-2000ng, D7-3000ng, D8-2000ng. Rest of volume: 2.5µL 10x buffer, 1µL E, 1µL P, filled to 25µL with water
Exceptions: D4, D8 had no E or P
Products:

Sample LabelDescriptionSource LabelQuantity
LP 9/4 D1, D2, D3MP2 digested with E&P at various DNA concentrationsMP2 8/25 KKB3
LP 9/4 D5, D6, D7MP3 digested with E&P at various DNA concentrationsMP3 8/25 KKB3
LP 9/4 D4, D8Negative controls (No E or P)MP2, MP31

Notes:Well: 1 - D1, 2 - D2, 3- D3, 4-D4, 5-Ladder, 6-empty, 7-D5, 8-D6, 9-D7, 10-D8
Stop Time: 3pm
Next: Figure out why PCR failed

Date: 9/9/2015
People in lab: Kent Gorday
PCR amplificaton of ocimene synthase, BCs of pGEX4T-1
Start Time: 2pm
Purpose: amplify ocimene synthase
Protocol: LM ed.2, PCRs: A1 - 10.5 water, 12.5 TaqMM, 0.5 VF2, 0.5 OcimeneR, 1µL 10^5 diluted ocimene gblock. Digest: D1 - 20 water, 2.5µL tango, 1 E, 1P 0.5 A1
Exceptions: PCR program: 95C 30s, 32 cycles (95C 20s, 57C 20s, 68C 2min), 68 5min, 4 hold
Products:

WellSample
45µL ladder
510µL 9/9 A1

Sample LabelDescriptionSource LabelQuantity
9/9 A1amplified ocimene synthasePart 3glock1
9/9 D1nothing9/9 A11
9/9 BC1pGEX4t-1 in amppGEX4t-13
9/9 A2amplified ocimene synthasepart3 gblock1

Results:
gel electrophoresis
Notes: Placed in red freezer box 8:00pm LP 9/9
Stop Time: 6pm
Next: run gel of A2

Date: 9/9/2015
People in lab: Kent Gorday
Gel of ocimene synthase PCR
Start Time: 8pm
Purpose: verify PCR product
Protocol: LM Ed2
Products:

WellSample
5ladder
610µL 9/9 A2

Results:
gel electrophoresis
Stop Time: 9:30pm
Next:

Date: 9/10/2015
People in lab: Levi Palmer
PCR of ocimene synthase gblock
Start Time: 5:30pm
Purpose: to amplify gblock for digestion
Protocol: LM ed2, A1 - 10 water, 12.5 MM, 0.5 VF2, 0.5 ocimeneR, 1.5 gblock; A2 - 9 water, 12.5 MM, 0.5 VF2, 0.5 ocimeneR, 2.5 µL gblock
Exceptions: ran with igem/hmp pcr program
Products:

Sample LabelDescriptionSource Label
A1 9/10 LPPCR amplified ocimene gblockocimene gblock
A2 9/10 LPPCR amplified ocimene gblockocimene gblock

Stop Time: 6pm
Next: Gel to confirm

Date: 9/10/2015
People in lab: Kira Buckowing
Gel of ocimene pcr
Start Time: 8:15pm
Purpose: confirm ocimene was amplified
Protocol: LM ed2
Products:

WellSample
3A1 LP 9/10
4ladder
5A2 LP 9/10

Results:
gel electrophoresis
Stop Time: 9:10pm
Next: looks fine, new stuff again

Date: 9/11/2015
People in lab: Levi Palmer
Final digest of ocimene block and bb
Start Time: 7:40pm
Purpose: to prepare ocimene for ligation
Protocol: lm ed2
Exceptions: 13µL final volume(buffer adjusted accordingly)
Products:

Sample LabelDescriptionSource Label
9/11 D1 LPocimene digested E&Pocimene gblock
9/11 D2 LPchlor bb digested E&PPSB1C3 2015 linear

Stop Time:
Next: Ligation

Date: 9/11/2015
People in lab: Levi Palmer
Final ligation of ocimene
Start Time: continued from prev
Purpose: to ligate ocimene into chlorbb
Protocol: LM ed2.
Exceptions: 1:1, 1:3, 1:5 molar ratios BB:Insert used
Products:

Sample LabelDescriptionSource Label
9/11 LP L11:1 chlorbb:ocimene9/11 LP D1, D2
9/11 LP L21:3 chlorbb:ocimene9/11 LP D1, D2
9/11 LP L31:5 chlorbb:ocimene9/11 LP D1, D2

Stop Time: 10:20pm
Next: transform products and/or pcr

Date: 9/12/2015
People in lab: Levi Palmer
PCR of ligations and gel
Start Time: 11am
Purpose: to check for complete oci+bb plasmids in ligations
Protocol: LM ed2
Exceptions: 0.3 µL of ligations used
Products:

WellSample
2A1
3A2
4ladder
5A3

Sample LabelDescriptionSource Label
LP 9/12 A1pcr of L19/11 L1
LP 9/12 A2pcr of L29/11 L2
LP 9/12 A3pcr of L39/11 L3

Results:
gel electrophoresis
Stop Time:
Next: gel extraction

Date: 9/12/2015
People in lab: Levi Palmer
GE of ocimene synthase, PCR, gel
Start Time: cont from prev
Purpose: To purify pcrs from gel and amplify and confirm
Protocol: IBI GE procedure, LM ed2
Exceptions: A4 - 2µL GE used, A5 - 4, A6 - 8, A7 - 4. A7 neg control, no primer
Products:

WellSample
2A4
3A5
4ladder
5A6
6A7

Sample LabelDescriptionSource LabelQuantity
LP GE1 9/12combined, gel purified ocimene synthase dnaA1, A2, A3 9/12 LP1
LP A4, A5, A6amplified ocimene synthaseGE1 LP 9/123
LP 9/12 A7neg control, no primerGE1 LP 9/121

Results: extensive smearing
Notes: GE has high salt conc.
Stop Time:
Next: Retry PCR of L1,2,3

Date: 9/12/2015
People in lab: Levi Palmer
PCR of GE1 and L1 L2 L3, pcr purification of A9
Start Time: cont from prev
Purpose: To amplify ocimene synthase, IBI PCR purification protocol
Protocol: LM ed2
Exceptions: A8 - 1µL L1, A9 - 5/5µL L2/L3, A10 - 2µL L2, A11 - 1 L2
Products:

WellSample
3A11
4ladder
5A8
6A9
7A10

Sample LabelDescriptionSource Label
PCR Pure LP 9/12ocimene synthase pcr amplified productLP 9/12 A9

Results: smearing
Stop Time:
Next: PCR

Date: 9/12/2015
People in lab: Levi Palmer
PCR of L1, L2, L3 and pcr pure 9/12 and dilution of primers, gel
Start Time: cont. from prev
Purpose: To amplify ocimene synthase
Protocol: VF2 - 0.16 mg+2.56 ml water, VR - 0.17 mg + 2.78ml water. LM ed2
Exceptions: A12 - 0.3 µL L2, A13 - 0.6 µL L2, A14 - 0.3µL L2, A15-0.3µL PCR Pure. A13 and A14 are double volume(MM adjusted accordingly)
Products:

WellSample
1A12
2A13
3A14
4ladder
5A15

Sample LabelDescriptionSource LabelQuantity
LP 9/12 A12,13,14amplified ocimene synthaseL2 LP 9/123
LP 9/12 A15amplified ocimene synthasePCR pure LP 9/121
VF2 LP 9/1510uM VF2 primerIDT VF22
VR LP 9/1510uM VR primerIDT VR2

Results:
gel electrophoresis
Notes: primers in both red and clear freezer boxes
Stop Time: 1:30 am
Next: Final PCR attempt, CT of L1, L2, L3

Date: 9/12/2015
People in lab: Levi Palmer
Final PCR of ocimene synthase
Start Time: cont. from prev
Purpose: Final PCR of ocimene synthase to eliminate smearing
Protocol: LM ED2
Exceptions: ALC - 0.2 µL L1 used
Products:

Sample LabelDescriptionSource Label
ALC LP 9/12amplified ocimene synthaseL1 9/12 LP

Sample LabelDescriptionSource LabelQuantity
9/13 CT1,4,5ocimene synthase cells?L1 LP 9/116
CT2 9/13ocimene synthase cells?L2 LP 9/112
CT3 9/13ocimene synthase cells?L3 LP 9/112
BC1,2,3,4,53µL CT broth in LB+chlorCT1,2,3,4,55

Stop Time: 2:30am
Next: gel

Date: 9/13/2015
People in lab: Kira Buckowing
CT of L1-3 9/12 LP
Start Time: 4:30pm
Purpose: To check for complete plasmids
Protocol: NEB chemical comp protocol
Exceptions: Used chemical comp cells
Stop Time: 7:20pm
Next: Hope for miracle

Date: 9/14/2015
People in lab: Kira Buckowing
BCs of CTs
Start Time: 5pm
Purpose: Grow cultures for MPs
Protocol: LM ed2
Products:

Sample LabelDescriptionSource LabelQuantity
BC 1, 2, 3, 4, 5BC of ocimene synthase?CT25
BC 6, 7, 8, 9, 10, 11ocimene synthase?CT35
BC 12-21ocimene synthase?CT510

Stop Time: 5:50pm
Next: miniprep anything that grows

Date: 9/15/2015
People in lab: Kira Buckowing
Minipreps
Start Time: 1:30pm
Purpose: Purify a plasmid of ocimene synthase
Protocol: Kitless miniprep procedure
Products:

Sample LabelDescriptionSource LabelQuantity
MP1-15is it ocimene synthase?BC1-1515

Stop Time: 7:30pm
Next: Digest with e&p, run gel

Date: 9/15/2015
People in lab: Kira Buckowing
Digest and gel
Start Time: 7:50pm
Purpose: to check for ocimene synthase (1750bp)
Protocol: LM ed2
Products:

WellSample
1D1/D9
2D2/D10
3D3/D11
4D4/D12
5L/L
6D5/D13
7D6/D14
8D7/D15

Sample LabelDescriptionSource LabelQuantity
KKB D1-15 9/15Ocimene synthase cut E&P?MP1-15 9/15 KKB15

Results:
gel electrophoresis
gel electrophoresis
Stop Time: 9:40PM
Next: This is the end.

Missouri cave photo by Lynn Dieter