Team:SDU-Denmark/labnotes9

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(Control restriction digest of cPCR product of PS in pSB1C3 and pSB1AK3)
(Colony PCR of FlhDCmut in pSB1AK3 and pSB1C3)
 
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== Flagella Group ==
== Flagella Group ==
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=== Two-step PCR of miniprep ===
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<br>
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==== First step: PCR of miniprep with mutation primers ====
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<br>
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''Done by:'' Louise <br>
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''Date:'' September 7th <br>
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''Protocol:'' [[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]] <br>
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''Notes:'' <br>
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'''3 x Premix 1:''' <br>
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114.3ul water <br>
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15ul Pfu Buffer <br>
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4.5ul dNTP <br>
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3ul MgSO4 <br>
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4.5ul FlhDC fw <br>
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4.5ul FlhDCmut rev <br>
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1.2ul PFU <br>
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3ul template (miniprep) <br><br>
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'''3 x Premix 2:''' <br>
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114.3ul water <br>
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15ul Pfu Buffer <br>
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4.5ul dNTP <br>
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3ul MgSO4 <br>
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4.5ul FlhDCmut fw <br>
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4.5ul FlhDC rev <br>
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1.2ul PFU <br>
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3ul template (miniprep) <br><br>
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''Results:''
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<br>
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NanoDrop: <br>
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'''Sample 1.1:''' Concentration: 359.5ng/ul Purity: 1.83/2.20 <br>
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'''Sample 2.1:''' Concentration: 304.1ng/ul Purity: 1.85/2.23 <br>
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<br>
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The PCR samples were run on a 1.5% gel with a 100bp-1000bp ladder. The first three samples are all positioned between 100bp and 200bp, this is the part of the gene run with FlhDCmut fv and FlhDC rev primers. The other three samples are all positioned betveen 900bp and 1000bp, this is the pcr with FlhDC fv and FlhDCmut rev primers. All samples looked okay and were pooled as sample 1.1 (light band) and sample 2.1 (heavy band)<br>
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[[Image:Team SDU-Denmark-Pfx pcr af miniprep.jpg|300px]]
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<br>
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A gel extraction of the PCR product of sample 1.1 and 2.1 were made:
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<br>
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[[Image:Team SDU-Denmark Gel extraction af FlhDCmut dele.jpg|300px]]
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<br>
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The gel extraction products were used in the preceding PCRs.
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==== Second step: 2-step PCR with mutated template sample 1.1 and 2.1 and FlhDC fw and rev primers ====
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<br>
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''Done by:'' Maria <br>
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''Date:'' September 9th <br>
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''Protocol:'' [[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]] <br>
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''Notes:'' <br>
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'''Premix:''' <br>
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38ul water <br>
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5ul PFU buffer + MgSO4 <br>
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1.5ul dNTP <br>
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1ul Sample 1.1 <br>
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1ul Sample 2.1 <br>
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1.5ul FlhDC fw primer <br>
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1.5ul FlhDC rev primer <br>
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0.5ul PFU <br><br>
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'''PCR Program:''' <br>
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<table style="text-align: left; height: 260px; width: 225px;" border="1"
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cellpadding="2" cellspacing="2">
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<tr>
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<td style="vertical-align: top; width: 102px;">1:Start<br>
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</td>
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<td style="vertical-align: top; width: 52px;">95C<br>
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</td>
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<td style="vertical-align: top; width: 45px;">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; width: 102px;">2: Denaturing<br>
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</td>
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<td style="vertical-align: top; width: 52px;">95C<br>
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</td>
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<td style="vertical-align: top; width: 45px;">30 sec<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; width: 102px;">3: Annealing<br>
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</td>
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<td style="vertical-align: top; width: 52px;">56C<br>
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</td>
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<td style="vertical-align: top; width: 45px;">30 sec<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; width: 102px;">4: Elongation<br>
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</td>
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<td style="vertical-align: top; width: 52px;">72C<br>
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</td>
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<td style="vertical-align: top; width: 45px;">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; width: 102px;">5:<br>
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</td>
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<td style="vertical-align: top; width: 52px;">GO TO<br>
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</td>
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<td style="vertical-align: top; width: 45px;">2 rep. 4x<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; width: 102px;">6: Denaturing<br>
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</td>
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<td style="vertical-align: top; width: 52px;">95C<br>
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</td>
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<td style="vertical-align: top; width: 45px;">30 sec<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; width: 102px;">7: Annealing<br>
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</td>
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<td style="vertical-align: top; width: 52px;">63C<br>
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</td>
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<td style="vertical-align: top; width: 45px;">30 sec<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; width: 102px;">8: Elongation<br>
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</td>
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<td style="vertical-align: top; width: 52px;">72C<br>
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</td>
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<td style="vertical-align: top; width: 45px;">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; width: 102px;">9:<br>
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</td>
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<td style="vertical-align: top; width: 52px;">GO TO<br>
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</td>
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<td style="vertical-align: top; width: 45px;">6 rep. 25x<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; width: 102px;">10: End <br>
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</td>
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<td style="vertical-align: top; width: 52px;">72C<br>
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</td>
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<td style="vertical-align: top; width: 45px;">5 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; width: 102px;">12: Hold<br>
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</td>
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<td style="vertical-align: top; width: 52px;">4C<br>
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</td>
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<td style="vertical-align: top; width: 45px;"><br>
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</td>
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</tr>
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</table> <br><br>
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''Results:'' <br>
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Some of the PCR product was run on a 1.5% gel with a 10kb ladder. <br>
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[[Image:Team SDU-Denmark God pcr af FlhDCmut (maria) 1.jpg|300px]]
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<br>
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The rest of the product was extracted from a new gel, unfortunately there were problems with the camera, so there is no picture of the gel extraction.
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<br>
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===== PCR of Gel extraction =====
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<br>
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''Done by:'' Maria <br>
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''Date:'' September 9th <br>
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''Protocol:'' [[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]] <br>
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''Notes:'' <br>
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'''Premix x 6:''' <br>
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234ul water <br>
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30ul PFU buffer + MgSO4 <br>
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9ul dNTP <br>
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9ul FlhDC fw primer <br>
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9ul FlhDC rev primer <br>
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2.5ul PFU <br>
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6ul template <br>
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<table style="text-align: left; height: 260px; width: 225px;" border="1"
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cellpadding="2" cellspacing="2">
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<tr>
 +
<td style="vertical-align: top; width: 102px;">1:Start<br>
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</td>
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<td style="vertical-align: top; width: 52px;">95C<br>
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</td>
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<td style="vertical-align: top; width: 45px;">2 min<br>
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</td>
 +
</tr>
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<tr>
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<td style="vertical-align: top; width: 102px;">2: Denaturing<br>
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</td>
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<td style="vertical-align: top; width: 52px;">95C<br>
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</td>
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<td style="vertical-align: top; width: 45px;">30sec<br>
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</td>
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</tr>
 +
<tr>
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<td style="vertical-align: top; width: 102px;">3: Annealing<br>
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</td>
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<td style="vertical-align: top; width: 52px;">63C<br>
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</td>
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<td style="vertical-align: top; width: 45px;">30 sec<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; width: 102px;">4: Elongation<br>
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</td>
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<td style="vertical-align: top; width: 52px;">72C<br>
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</td>
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<td style="vertical-align: top; width: 45px;">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; width: 102px;">5:<br>
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</td>
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<td style="vertical-align: top; width: 52px;">GO TO<br>
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</td>
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<td style="vertical-align: top; width: 45px;">2 rep. 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; width: 102px;">6: End <br>
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</td>
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<td style="vertical-align: top; width: 52px;">72C<br>
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</td>
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<td style="vertical-align: top; width: 45px;">5 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; width: 102px;">7: Hold<br>
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</td>
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<td style="vertical-align: top; width: 52px;">4C<br>
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</td>
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<td style="vertical-align: top; width: 45px;"><br>
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</td>
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</tr>
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</table> <br><br>
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<br>
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<br>
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''Results:''
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<br>
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The PCR worked fine, unfortunately there were problems with the camera, so there is no picture of the gel. <br>
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=== Restriction Digest of Gel Extracted FlhDCmut, pSB1C3 and pSB1AK3 ===
=== Restriction Digest of Gel Extracted FlhDCmut, pSB1C3 and pSB1AK3 ===
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'''Results:''' <Br>
'''Results:''' <Br>
<Br><Br>
<Br><Br>
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=== Colony PCR of FlhDCmut in pSB1AK3 and pSB1C3 ===
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<br>
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'''Date:''' 9/15 2010<Br>
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'''Done By:''' Louise<Br>
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'''Protocol:''' [https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.3 CP1.3]<Br>
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'''Notes:''' We made 8 C-PCRs with colonies transformed with pSB1AK3 and 7 C-PCRs with colonies transformed with pSB1C3 (we made 8 but due to a mishab sample 5 was not run. Taq polymerase, VF2- and VR primers were used. Only Elongation time was altered from 2 min in the protocol to 1.5 min<Br>
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'''Results:''' <Br>
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The picture first picture is of FlhDCmut in pSB1AK3. This shows one band at about 400bp, the double terminator. This indicates that non of the colonies had the right plasmid.<br>
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The second picture is of FlhDCmut in pSB1C3.This shows the 4 of the 7 colonies had plasmids containing the right incert (sample 1,3, 7 and 8). These samples show a band at approximately 1200bp (FlhDCmut in pSB1C3 is 1248bp). the three other samples show a band heavier than 2000bp <br>
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[[Image:Team SDU-Denmark FlhDCmut indsat 1. gang i 1AK3 og 1C3.jpg|300px]]
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<br>
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----
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=== Colony PCR of FlhDCmut in pSC1AK3 ===
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<br>
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'''Date:''' 9/15 2010<Br>
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'''Done By:''' Louise<Br>
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'''Protocol:''' [https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.3 CP1.3]<Br>
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'''Notes:''' We made 8 C-PCRs with colonies transformed with pSB1AK3 and 7 C-PCRs with colonies transformed with pSB1C3 (we made 8 but due to a mishab sample 5 was not run. Taq polymerase, VF2- and VR primers were used. Only Elongation time was altered from 2 min in the protocol to 1.5 min<Br>
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'''Results:''' <Br>
== Photosensor group ==
== Photosensor group ==
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<br>
<br>
Results: Only sample C showed bands at the correct length (around 2180 BP), where as all other bands were at the length of a double terminator (b0015) without the photosensor insert. All samples were properly cut indicating the presence of biobrick pre- and suffix in these. Sample C will be miniprepped and sent off to sequencing. <br>
Results: Only sample C showed bands at the correct length (around 2180 BP), where as all other bands were at the length of a double terminator (b0015) without the photosensor insert. All samples were properly cut indicating the presence of biobrick pre- and suffix in these. Sample C will be miniprepped and sent off to sequencing. <br>
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[[Image:Team-SDU-Denmark-RDPS2.jpg|400px]]
<br>
<br>
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<br>
<br>
Results: Samples K (former C) and Q produced bands at the right length of around 2150 BP, which eans that sample Q will also be miniprepped and send to sequencing together with the previously chosen sample C (now K).<br>
Results: Samples K (former C) and Q produced bands at the right length of around 2150 BP, which eans that sample Q will also be miniprepped and send to sequencing together with the previously chosen sample C (now K).<br>
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[[Image:Team-SDU-Denmark-CPCRBST.jpg|400px]]
<br>
<br>
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<br>
<br>
Sample Q did not fulfill DNA technologies sample specifications for sequencing we waited with sending it in, since we were expecting the results from sample C which was sent in earlier. Since those returned positive, sample Q was never sent in, since we just proceeded with sample C.
Sample Q did not fulfill DNA technologies sample specifications for sequencing we waited with sending it in, since we were expecting the results from sample C which was sent in earlier. Since those returned positive, sample Q was never sent in, since we just proceeded with sample C.
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<br><br>
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<br>
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[[Image:Team-SDU-Denmark-MINIPST.jpg|400px]]<br>

Latest revision as of 10:23, 1 October 2010