Team:SDU-Denmark/labnotes14

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Protocols: LG1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#LG1.1]<br>
Protocols: LG1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#LG1.1]<br>
Notes: <br>
Notes: <br>
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For each of the ligations three ligation mixtures were prepared. vector concentrations of 41,6ng/uL (PSB1C3) was used for mixture. Appropiate amount of insert was added to reach vector:insert ratios of 1:1, 1:2 and 1:3 respectively. <br>
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Ligation mixtures (B0015 in pSB3C5):<br>
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<table style="text-align: left;" border="1"
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cellpadding="2" cellspacing="2">
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    <tr>
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      <td></td>
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      <td>L1</td>
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      <td>L2</td>
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      <td>L3</td>
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    </tr>
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    <tr>
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      <td>T4 ligase buffer</td>
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      <td>2uL</td>
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      <td>2uL</td>
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      <td>2uL</td>
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    </tr>
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    <tr>
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      <td>T4 ligase</td>
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      <td>1uL</td>
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      <td>1uL</td>
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      <td>1uL</td>
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    </tr>
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    <tr>
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      <td>pSB1C3</td>
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      <td>1uL</td>
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      <td>1uL</td>
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      <td>1uL</td>
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    </tr>
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    <tr>
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      <td>K343002 </td>
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      <td>0.65uL</td>
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      <td>1.30uL</td>
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      <td>1.96uL</td>
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    </tr>
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    <tr>
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      <td>H<small>2</small>0</td>
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      <td>34.35uL</td>
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      <td>33,70uL</td>
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      <td>33.04uL</td>
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    </tr>
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</table><br>
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The samples was incubated at 17C ON at used for transformation<br>
=== Transformations of K343002 in PSB1C3 into top10 E. Coli ===
=== Transformations of K343002 in PSB1C3 into top10 E. Coli ===
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</tr>
</tr>
</table>
</table>
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=== Coloni PCR on transformations of K343002 in PSB1C3 ===
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Date: 30/09<br>
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Done by: Tommy<br>
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Methods: PCR<br>
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Protocols: CP1.3[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.3]<br>
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Notes: <br>
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Because the first coloni PCR of the transformants didn't give us the the right pieces 32 new colonies where chosen:<br>
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1-16: ninaB in PSB1C3<br>
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VF2 and VR used as primers in the TAQ PCR:<br>
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PCR Program:<br>
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<table style="text-align: left;" border="1" cellpadding="2"
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cellspacing="2">
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<tr>
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<td style="vertical-align: top;">Start<br>
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</td>
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<td style="vertical-align: top;">94&nbsp; C<br>
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</td>
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<td style="vertical-align: top;">2 min<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top;">Denaturing<br>
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</td>
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<td style="vertical-align: top;">94 C<br>
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</td>
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<td style="vertical-align: top;">1 min<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top;">Annealing<br>
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</td>
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<td style="vertical-align: top;">55 C<br>
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</td>
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<td style="vertical-align: top;">1 min<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top;">Elongation<br>
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</td>
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<td style="vertical-align: top;">72 C<br>
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</td>
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<td style="vertical-align: top;">2:30 min<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top;">Goto2<br>
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</td>
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<td style="vertical-align: top;">rep<br>
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</td>
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<td style="vertical-align: top;">29x<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top;">End<br>
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</td>
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<td style="vertical-align: top;">72 C<br>
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</td>
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<td style="vertical-align: top;">2:30 min<br>
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</td>
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</tr>
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<tr>
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<td style="vertical-align: top;">Hold<br>
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</td>
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<td style="vertical-align: top;">4 C<br>
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</td>
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<td style="vertical-align: top;"><br>
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</td>
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</tr>
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</table>
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Colonies 1, 2, 3 and 4 was selected for minipreps and freezing cultures.

Latest revision as of 16:32, 16 October 2010

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