Team:Alberta/Notebook June

From 2010.igem.org

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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="https://2010.igem.org/wiki/index.php?title=Team:Alberta/Notebook_May" onclick="" return false;">11-05-2010</a> with the following recipe:</p>
<p>To optimize the Restriction and ligation of BsaI-HF, digested 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 false;">11-05-2010</a> with the following recipe:</p>
<p>Digestion Recipe:<br>
<p>Digestion Recipe:<br>
-
<ul>14&mu;L either A/B' or B/A' Kanamycin resistance cassette (approx. 100ng/&mu;L)
+
<ul>14&mu;L either A/B' or B/A' Kanamycin resistance cassette (approx. 100ng/&mu;L)</ul>
-
<ul>5&mu;L 1/10 dillution of 100X BSA
+
<ul>5&mu;L 1/10 dillution of 100X BSA</ul>
-
<ul>5&mu;L 10X NEBuffer4  
+
<ul>5&mu;L 10X NEBuffer4</ul>
-
<ul>1.5&mu:L BsaI-HF
+
<ul>1.5&mu:L BsaI-HF</ul>
-
<ul>24.5&mu;L MilliQ
+
<ul>24.5&mu;L MilliQ</ul></p>
<p>Digested at 50<sup>o</sup>C for 1hour, heat inactivated the enzyme at 65<sup>o</sup>C for 20 minutes
<p>Digested at 50<sup>o</sup>C for 1hour, heat inactivated the enzyme at 65<sup>o</sup>C for 20 minutes
PCR purified the digests.</p>
PCR purified the digests.</p>
 +
</div>
 +
 +
<div align="left" id='div3' class="hideme">
 +
<p>June 3, 2010</p><br>
 +
<span class="h2">Building Parts</span>
 +
<p>Tried to ligate the KanA/B' fragments to itself.  Tried to ligate the KanB/A' fragments to itself.  Tried to ligate the KanA/B' fragments to the KanB/A' fragments.</p>
 +
<p>Ligation Recipe:<br>
 +
<ul>8&mu;L of digest from <a style="color: #fff200" href="" onclick="return toggleMe('div2')" return false;">02-06-2010</a> (either 8&mu;L of one of the fragments or 4&mu;L of each)</ul>
 +
<ul>1&mu;L T4 DNA ligase</ul>
 +
<ul>6&mu;L 5X Buffer</ul>
 +
<ul>15&mu;L MilliQ H<sub>2</sub>O</ul></p>
 +
<p>Took aliquots of ligations at varying times and ran 1% agarose gels to test ligation.</p> 
 +
<!--images-->
 +
<p>Digested some of Minipreps of the KanA/B' fragment inserted into pSB1C3 from <a style="color: #fff200" href="" onclick="return toggleMe('div2')" return false;">02-06-2010</a>.</p>
 +
<p>Digestion Recipe:<br>
 +
<ul>14&mu;L plasmid (approx 300-400ng/&mu:L)</ul>
 +
<ul>5&mu;L 10X NEBuffer 4</ul>
 +
<ul>5&mu;L 1/10 dilution of 100X BSA</ul>
 +
<ul>1.5&mu;L BsaI-HF</ul>
 +
<ul>24.5&mu;L MilliQ H<sub>2</sub>O</ul></p>
 +
 +
<p>Made a 1/100 dilution of AmpR and TetR PCR Products from [[#27-05-2010|27-05-2010]] and performed PCR reactions to produce antibiotic inserts to make parts.</p>
 +
<p>PCR Recipe:<br>
 +
<ul>35.5&mu;L MilliQ H<sub>2</sub>O</ul>
 +
<ul>1&mu;L 10 uM dNTPs</ul>
 +
<ul>5&mu;L 10X PCR Buffer</ul>
 +
<ul>2&mu;L 50 uM MgCl<sub>2</sub></ul>
 +
<ul>2.5&mu;L Primer + (ApR 1/10+ or TR 1/10+)</ul>
 +
<ul>2.5&mu;L Primer - (ApR 1/10- or TR 1/10-)</ul>
 +
<ul>1&mu;L Template (1/100 diluted AmpR or TetR)</ul>
 +
<ul>0.5&mu;L Taq Polymerase</ul></p>
 +
<p>PCR Program:<br>
 +
<ul>1.  5 min-94<sup>o</sup>C</ul>
 +
<ul>2.  45 sec-94<sup>o</sup>C</ul>
 +
<ul>3.  1 min-60<sup>o</sup>C</ul>
 +
<ul>4.  1 min-72<sup>o</sup>C</ul>
 +
<ul>5.  Repeat 2 through 4 35 times</ul>
 +
<ul>6.  5 min-72<sup>o</sup>C</ul></p>
</div>
</div>

Revision as of 17:52, 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.

June 3, 2010


Building Parts

Tried to ligate the KanA/B' fragments to itself. Tried to ligate the KanB/A' fragments to itself. Tried to ligate the KanA/B' fragments to the KanB/A' fragments.

Ligation Recipe:

    8μL of digest from 02-06-2010 (either 8μL of one of the fragments or 4μL of each)
    1μL T4 DNA ligase
    6μL 5X Buffer
    15μL MilliQ H2O

Took aliquots of ligations at varying times and ran 1% agarose gels to test ligation.

Digested some of Minipreps of the KanA/B' fragment inserted into pSB1C3 from 02-06-2010.

Digestion Recipe:

    14μL plasmid (approx 300-400ng/&mu:L)
    5μL 10X NEBuffer 4
    5μL 1/10 dilution of 100X BSA
    1.5μL BsaI-HF
    24.5μL MilliQ H2O

Made a 1/100 dilution of AmpR and TetR PCR Products from [[#27-05-2010|27-05-2010]] and performed PCR reactions to produce antibiotic inserts to make parts.

PCR Recipe:

    35.5μL MilliQ H2O
    1μL 10 uM dNTPs
    5μL 10X PCR Buffer
    2μL 50 uM MgCl2
    2.5μL Primer + (ApR 1/10+ or TR 1/10+)
    2.5μL Primer - (ApR 1/10- or TR 1/10-)
    1μL Template (1/100 diluted AmpR or TetR)
    0.5μL Taq Polymerase

PCR Program:

    1. 5 min-94oC
    2. 45 sec-94oC
    3. 1 min-60oC
    4. 1 min-72oC
    5. Repeat 2 through 4 35 times
    6. 5 min-72oC

Retrieved from "http://2010.igem.org/Team:Alberta/Notebook_June"