Team:Alberta/Notebook June

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<h2 class="calendar">May 2010</h2>
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<h2 class="calendar">June 2010</h2>
    
    
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<div align="left" id='div30' class="hideme">
 
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<p>May 30, 2010</p><br>
 
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<span class="h2">Building Parts</span>
 
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<p>From the transformation of DH5&alpha; cells with pSB1C3-KanR performed on <a style="color: #fff200" href="" onclick="return toggleMe('div28')" return false;">28-05-2010</a>, we took 12 distinct colonies of each KanA/B' and KanB/A', streaked them on a new chloramphenicol plate and inoculated 5ml liquid cultures with the appropriate antibiotics overnight at 37<sup>o</sup>C. We also picked colonies of pSB4A5-J04450, pSB4C5-J04450 and pSB3T5-J04450, streaked and made 5mL liquid cultures of them too.</p>
 
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<div align="left" id='div1' class="hideme">
<div align="left" id='div1' class="hideme">
<p>June 1, 2010</p><br>
<p>June 1, 2010</p><br>
<span class="h2">Building Parts</span>
<span class="h2">Building Parts</span>
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<p>KanA/B' and KanB/A' fragements PCRed on <a style="color: #fff200" href="https://2010.igem.org/wiki/index.php?title=Team:Alberta/Notebook_May" onclick="return toggleMe('div11')" return false;">11-05-2010</a>, digested with BsaI-HF at 37<sup>o</sup>C for 1.5hours, heat inactivated at 65<sup>o</sup>C for 30 minutes.  Tried to ligate KanA/B' fragments to each other and tried to ligate KanB/A' fragments to each other.  Also tried to ligate KanA/B' fragments with KanB/A'.  Ligated with T4 DNA ligase for 3 hours at 21<sup>o</sup>C.
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<p>KanA/B' and KanB/A' fragements PCRed on <a style="color: #fff200" href="https://2010.igem.org/wiki/index.php?title=Team:Alberta/Notebook_May" onclick="return toggleMe('div11')" return false;">11-05-2010</a>, digested with BsaI-HF at 37<sup>o</sup>C for 1.5hours, heat inactivated at 65<sup>o</sup>C for 30 minutes.  Tried to ligate KanA/B' fragments to each other and tried to ligate KanB/A' fragments to each other.  Also tried to ligate KanA/B' fragments with KanB/A'.  Ligated with T4 DNA ligase for 3 hours at 21<sup>o</sup>C.</p>
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Set up liquid cultures of KanRA/B'-Bsa and KanR B/A'-Bsa in pSB1C3 from plates streaked with on <a style="color: #fff200" href="https://2010.igem.org/wiki/index.php?title=Team:Alberta/Notebook_May" onclick="return toggleMe('div30')" return false;">30-05-2010</a>.
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<p>Set up liquid cultures of KanRA/B'-Bsa and KanR B/A'-Bsa in pSB1C3 from plates streaked with on <a style="color: #fff200" href="https://2010.igem.org/wiki/index.php?title=Team:Alberta/Notebook_May" onclick="" return false;">30-05-2010</a></p>.
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<p>May 10, 2010</p><br>
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<p>June 2, 2010</p><br>
<span class="h2">Building Parts</span>
<span class="h2">Building Parts</span>
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<p>PCR a Kanamycin resistance cassette fragment from p1003 with primers containing either the A/B' ends or the B/A' ends. (Fragments formed called KanR A/B'-Bsa and KanR B/A'-Bsa respectively)</p>
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<p>Miniprepped liquid cultures from <a style="color: #fff200" href="" onclick="return toggleMe('div1')" return false;">01-06-2010</a>. Ran a 1% agarose gel of the ligations performed <a style="color: #fff200" href="" onclick="return toggleMe('div1')" return false;">01-06-2010</a>.</p>  
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<p>Recipe:</br>
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<!-- image of gel-->  
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<ul>1&mu;L p1003 (approx. 1ng)</ul>
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<p>To optimize the Restriction and ligation of BsaI-HF, digested KanA/B' and KanB/A' fragements PCRed on <a style="color: #fff200" href="" onclick="" return false;">11-05-2010</a> with the following recipe:</p>
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<ul>2.5&mu;L prA_p1003+</ul>
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<ul>2.5&mu;L prB'_p1003-</ul>
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<ul>5&mu;L 10X PCR buffer</ul>
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<ul>1&mu;L 10uM dNTPs</ul>
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<ul>2&mu;L 50uM MgCl<sub>2</sub></ul>
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<ul>0.5&mu;L Taq polymerase</ul>
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<ul>35.5&mu;L MilliQ H<sub>2</sub>O</ul></p>
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<p>Same recipe for KanR B/A'-Bsa except primers are prB_p1003+ and prA'_p1003-. </p>
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<p>Program:
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<ul>5 min-94<sup>o</sup>C</ul>
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<ul>45 sec-94<sup>o</sup>C</ul>
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<ul>1 min-60<sup>o</sup>C</ul>
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<ul>1 min-72<sup>o</sup>C</ul>
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<ul>Repeat 2 through 4 35 times</ul>
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<ul>5 min-72<sup>o</sup>C</ul></p>
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<!--Image of gel performed that day. 5&mu;L of each PCR reaction, 1&mu;L of 10X loading dye and 4&mu;L MilliQ water in a 1% agarose gel -->
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<div align="left" id='div11' class="hideme">
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<p>Digestion Recipe:<br>
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<p>May 11, 2010</p><br>
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<ul>14&mu;L either A/B' or B/A' Kanamycin resistance cassette (approx. 100ng/&mu;L)
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<span class="h2">Building Parts</span>
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<ul>5&mu;L 1/10 dillution of 100X BSA
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<p>PCR purification of KanR A/B'-Bsa and KanR B/A'-Bsa created <a style="color: #fff200" href="" onclick="return toggleMe('div10')" return false;">10-05-2010</a> with Qiagen PCR cleanup kit.</p>
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<ul>5&mu;L 10X NEBuffer4
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<p>Determined concentrations by nanodrop.  KanA/B'-Bsa: 101.1ng/&mu;L KanB/A'-Bsa:89.6ng/&mu;L</p>
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<ul>1.5&mu:L BsaI-HF
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<ul>24.5&mu;L MilliQ
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<p>Digested at 50<sup>o</sup>C for 1hour, heat inactivated the enzyme at 65<sup>o</sup>C for 20 minutes
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PCR purified the digests.</p>
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<p>May 17, 2010</p><br>
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<span class="h2">Building Parts</span>
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<p>Innoculated 250mL overnight cultures with 10mL,4mL and 2mL of a starter culture of DH5&alpha;.  Left shaking at 18<sup>o</sup>C overnight. </p>
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<p>May 18, 2010</p><br>
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<span class="h2">Building Parts</span>
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<p>Prepared DH5&alpha; E.Coli competent cells using the Inoue Method. </p>
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<p>Transformed DH5&alpha; cells with pSB1C3-J04450 and grew overnight at 37<sup>o</sup>C on Chloramphenicol plates</p>
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<p>May 19, 2010</p><br>
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<span class="h2">Building Parts</span>
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<p>From the transformation of DH5&alpha; cells with pSB1C3-J04450 performed on <a style="color: #fff200" href="" onclick="return toggleMe('div18')" return false;">18-05-2010</a>, we took 4 distinct colonies, streaked them on a new chloramphenicol plate and inoculated 5ml liquid cultures.</p>
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<p>May 20, 2010</p><br>
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<span class="h2">Building Parts</span>
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<p>Performed a Miniprep of 3 of the 4 5ml liquid cultures of DH5&alpha; cells with pSB1C3-J04450 from <a style="color: #fff200" href="" onclick="return toggleMe('div19')" return false;">19-05-2010</a>.  Took a 1&mu;L sample of the Miniprep solutions and digested with NotI at 37<sup>o</sup>C for 1 hour. </p>
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<p>Digestion Recipe:
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<ul>1&mu;L Miniprep (between 153.2 ng/&mu;l and 302.7ng/&mu;l determined by nanodrop)</ul>
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<ul>1&mu;L NotI</ul>
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<ul>1&mu;L 10X ReACT 3</ul>
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<ul>7&mu;L MilliQ</ul></p>
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<p>Ran Digestion on a 1% agarose gel to check that the plasmid obtained with what we expected. </p>
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<!-- Image of Gel-->
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<p>May 25, 2010</p><br>
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<span class="h2">Building Parts</span>
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<p>Made 1.5mL LB liquid cultures of pSB1C3 from the plate streaked on <a style="color: #fff200" href="" onclick="return toggleMe('div19')" return false;">19-05-2010</a> and added chloramphenicol.</p>
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<p>May 26, 2010</p><br>
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<span class="h2">Building Parts</span>
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<p>Made 3 glycerol stocks of pSB1C3 from overnight made <a style="color: #fff200" href="" onclick="return toggleMe('div25')" return false;">25-05-2010</a>.</p>
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</div>
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<div align="left" id='div27' class="hideme">
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<p>May 27, 2010</p><br>
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<span class="h2">Building Parts</span>
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<p>Digested both A/B' and B/A' Kanamycin Resistance cassettes fragments from <a style="color: #fff200" href="" onclick="return toggleMe('div11')" return false;">11-05-2010</a> and pSB1C3 from <a style="color: #fff200" href="" onclick="return toggleMe('div20')" return false;">20-05-2010</a> with NotI at 37<sup>o</sup>C for 1 hour. Heat inactivated the NotI for 10 minutes at 65<sup>o</sup>C.  Ligated the Kanamycin Resistance cassettes into pSB1C3 at 16<sup>o</sup>C for 1 hour then took 15&mu;L to room temperature for 2 hours.  Transformed 100&mu;L of DH5&alpha; cells with 5&mu;L of RT ligation reaction. Plated transformation on plates with both Chloramphenicol and Kanamycin.</p>
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<p>Digestion Recipe:
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<ul>1&mu;L Miniprep (302.7ng/&mu;l determined by nanodrop)</ul>
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<ul>2&mu;L either A/B' or B/A' Kanamycin resistance cassette (approx. 100ng/&mu;L)</ul>
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<ul>1&mu;L NotI</ul>
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<ul>1&mu;L 10X ReACT 3</ul>
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<ul>:5&mu;L MilliQ</ul></p>
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<p>Ligation Recipe:
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<ul>10&mu;L of Digest solution</ul>
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<ul>1&mu;L T4 DNA ligase</ul>
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<ul>6&mu;L 5X Buffer</ul>
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<ul>13&mu;L MilliQ H<sub>2</sub>O</ul></p>
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<p>Also transformed pSB4A5-J04450, pSB4C5-J04450 and pSB3T5-J04450 from the 2010 biobrick parts into DH5&alpha; cells.</p>
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<p>Performed PCR reactions to create parts with antibiotic resistance with negative controls.</p>
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<p>PCR Recipe:
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<ul>3&mu;L 10X PCR Buffer</ul>
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<ul>1&mu;L 10 uM dNTPs</ul>
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<ul>2&mu;L 50 uM MgCl<sub>2</sub></ul>
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<ul>17.5&mu;L MilliQ H<sub>2</sub>O</ul>
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<ul>0.5&mu;L Taq Polymerase</ul>
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<ul>1&mu;L Template (psB4A5-J04450, psB4C5-J04450 or psB3T5-J04450)</ul>
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<ul>2.5&mu;L Primer + (PrA psB4A5 ApR+, PrA psB4C5 ChR+ or PrA psB3T5 TR+)</ul>
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:2.5&mu;L Primer - (PrB psB4A5 ApR-, PrB psB4C5 ChR- or PrB psB3T5 TR-)</p>
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<p>PCR Program:
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<ul>5 min-94<sup>o</sup>C</ul>
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<ul>45 sec-94<sup>o</sup>C</ul>
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<ul>1 min-60<sup>o</sup>C</ul>
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<ul>1 min-72<sup>o</sup>C</ul>
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<ul>Repeat 2 through 4 35 times</ul>
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<ul>5 min-72<sup>o</sup>C</ul></p>
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<!--gel images of PCR Products (Alina's) and ligated and pre-digested samples (Jeremy's)-->
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<p>May 28, 2010</p><br>
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<span class="h2">Building Parts</span>
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<p>We got colonies!! (it's a fantastic feeling) We then lovingly put them in the cold room to await our return from Calgary</p>
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Revision as of 16:12, 12 July 2010

genomikon


June 2010

  • Su
  • Mo
  • Tu
  • We
  • Th
  • Fr
  • Sa
iGEM 2010 Notebook

The lab notebook chronicles our journey in the creation of the Genomikon kit. Many paths were woven together in space and time to reach this finished masterpiece. To help you navigate through these trials with us we have laid out our notebook in a layered fashion. This page gives a sketch of each project and how it interacts with each other. Then follow the links to a projects page for time line of the major landmarks and accomplishments. If you require more details on the project the links within that page will take you to our day-by-day work log.

Building Parts

The Building Parts project was responsible to first build a plasmid (plasmid 01)that contained our own specialized prefix and suffix nested inside of the standard BioBrick prefix and suffix. After plasmid 01 existed we inserted the CcdB gene (the "death" gene) between our prefix and suffix removing the gene for Kanamycin resistance (plasmid 02). Plasmid 02 is fantastic base plasmid from which we are able to amplify any part at all because it provides a positive selection marker when transformed into DH5α. At this point we were able to make parts en masse to put in our kit. After obtaining a particular part in a plasmid we PCRed the part and digested it ready to use in Assembly or to Test the plasmid.

Testing Parts

Before we were able to test parts we created 2 base testing plasmids (vector 01 and vector 02). Vector 01 is designed to test Open Reading Frame parts, or parts that code for proteins. The part is flanked by a promoter and the start codon on one side and a stop codon and terminator on the other. Vector 02 is designed to test linker parts, or parts that control the expression of the Open Reading Frame parts they are next two. In Vector 02 the part is flanked by two distinct reporter genes, that by comparing the relative expression of the 2 reporter genes we can determine the behavior of the linking part.

Assembly Method

Insert description here.

Plates

Insert description here.

Competent Cells

Insert description here.

Software

Insert description here.

June 1, 2010


Building Parts

KanA/B' and KanB/A' fragements PCRed on 11-05-2010, digested with BsaI-HF at 37oC for 1.5hours, heat inactivated at 65oC for 30 minutes. Tried to ligate KanA/B' fragments to each other and tried to ligate KanB/A' fragments to each other. Also tried to ligate KanA/B' fragments with KanB/A'. Ligated with T4 DNA ligase for 3 hours at 21oC.

Set up liquid cultures of KanRA/B'-Bsa and KanR B/A'-Bsa in pSB1C3 from plates streaked with on 30-05-2010

.

June 2, 2010


Building Parts

Miniprepped liquid cultures from 01-06-2010. Ran a 1% agarose gel of the ligations performed 01-06-2010.

To optimize the Restriction and ligation of BsaI-HF, digested KanA/B' and KanB/A' fragements PCRed on 11-05-2010 with the following recipe:

Digestion Recipe:

    14μL either A/B' or B/A' Kanamycin resistance cassette (approx. 100ng/μL)
      5μL 1/10 dillution of 100X BSA
        5μL 10X NEBuffer4
          1.5&mu:L BsaI-HF
            24.5μL MilliQ

            Digested at 50oC for 1hour, heat inactivated the enzyme at 65oC for 20 minutes PCR purified the digests.

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