Team:Exeter/lab book/novpol/wk9
From 2012.igem.org
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<!------------ENTER LAB BOOK HERE: PLEASE INCLUDE DATES WITHIN THE WEEK------------> | <!------------ENTER LAB BOOK HERE: PLEASE INCLUDE DATES WITHIN THE WEEK------------> | ||
+ | |||
+ | |||
+ | <p>**<b>Monday 03/09/12</b>**</p><br> | ||
+ | <p><b>9.00am</p></b> | ||
+ | <p>Transformations using Gene_Terminator.</p> | ||
+ | <ul>* Tetr_RBS + Cyc_Term</ul> | ||
+ | <ul>* Tetr_RBS + HAS_Term </ul> | ||
+ | <ul>* ompA + sacB_Term </ul> | ||
+ | |||
+ | <TABLE BORDER="3" ALIGN="CENTRE" WIDTH="50%" CELLPADDING="4" CELLSPACING="3"> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>Sample</b></TH> | ||
+ | <TH><b>Conc of DNA (ng/μl)</b></TH> | ||
+ | <TH><b>DNA required (μl)</b></TH> | ||
+ | <TH><b>Water required (μl)</b></TH> | ||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>Cyc_Term 2</b></TH> | ||
+ | <TD>142.8</TD> | ||
+ | <TD>3.5</TD> | ||
+ | <TD>13.3</TD> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>HAS_Term 1</b></TH> | ||
+ | <TD>104.6</TD> | ||
+ | <TD>4.8</TD> | ||
+ | <TD>12.0</TD> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>SacB_Term 1</b></TH> | ||
+ | <TD>119.3</TD> | ||
+ | <TD>4.2</TD> | ||
+ | <TD>12.6</TD> | ||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>Kanamycin Plasmid</b></TH> | ||
+ | <TD>112.8</TD> | ||
+ | <TD>4.43</TD> | ||
+ | <TD>12.37</TD> | ||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>Tetr_RBS</b></TH> | ||
+ | <TD>42.2</TD> | ||
+ | <TD>11.85</TD> | ||
+ | <TD>4.95</TD> | ||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>OmpA</b></TH> | ||
+ | <TD>70.2</TD> | ||
+ | <TD>7.12</TD> | ||
+ | <TD>9.68</TD> | ||
+ | </TR> | ||
+ | </TABLE> | ||
+ | |||
+ | <TABLE BORDER="3" ALIGN="CENTRE" WIDTH="50%" CELLPADDING="4" CELLSPACING="3"> | ||
+ | |||
+ | <TR> | ||
+ | <TH COLSPAN="2"><BR><H4>Upstream</H4></TH> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>DNA</b></TH> | ||
+ | <TD>500ng</TD> | ||
+ | |||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>BUFFER (10x)</b></TH> | ||
+ | <TD>2μl</TD> | ||
+ | |||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>BSA (100x)</b></TH> | ||
+ | <TD>0.2μl</TD> | ||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>Water</b></TH> | ||
+ | <TD>up to 20μl total V</TD> | ||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>ENZYME 1 - XbaI</b></TH> | ||
+ | <TD>0.5μL</TD> | ||
+ | |||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>ENZYME 2 - PstI</b></TH> | ||
+ | <TD>0.5μL</TD> | ||
+ | </TR> | ||
+ | <TR> | ||
+ | <TH COLSPAN="2"><BR><H4>Downstream</H4></TH> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>DNA</b></TH> | ||
+ | <TD>500ng</TD> | ||
+ | |||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>BUFFER (10x)</b></TH> | ||
+ | <TD>2μl</TD> | ||
+ | |||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>BSA (100x)</b></TH> | ||
+ | <TD>0.2μl</TD> | ||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>Water</b></TH> | ||
+ | <TD>up to 20μl total V</TD> | ||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>ENZYME 1 - ECORI</b></TH> | ||
+ | <TD>0.5μL</TD> | ||
+ | |||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>ENZYME 2 - SpeI</b></TH> | ||
+ | <TD>0.5μL</TD> | ||
+ | </TR> | ||
+ | <TR> | ||
+ | <TH COLSPAN="2"><BR><H4>Kanamycin Plasmid</H4></TH> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>DNA</b></TH> | ||
+ | <TD>500ng</TD> | ||
+ | |||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>BUFFER (10x)</b></TH> | ||
+ | <TD>2μl</TD> | ||
+ | |||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>BSA (100x)</b></TH> | ||
+ | <TD>0.2μl</TD> | ||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>Water</b></TH> | ||
+ | <TD>up to 20μl total V</TD> | ||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>ENZYME 1 - ECORI</b></TH> | ||
+ | <TD>0.5μL</TD> | ||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>ENZYME 2 - PstI</b></TH> | ||
+ | <TD>0.5μL</TD> | ||
+ | </TR> | ||
+ | |||
+ | </TABLE> | ||
+ | |||
+ | <p>Plasmid + Upstream + Downstream</p> | ||
+ | <p>Plasmid + Tetr_RBS + Cyc_Term</p> | ||
+ | <p>Plasmid + Tetr_RBS + HAS_Term</p> | ||
+ | <p>Plasmid + OmpA + SacB_Term</p> | ||
+ | |||
+ | <p><b>11.20am</b></p> | ||
+ | <p>All in the incubator for digestion period. </p> | ||
+ | <p><b>12.40pm</b></p> | ||
+ | <p>Heat denatured enzymes at 80°C for 20 minutes.</p> | ||
+ | <p><b>1.00pm</b></p> | ||
+ | <p>Ligation process</p> | ||
+ | <TABLE BORDER="3" ALIGN="CENTRE" WIDTH="50%" CELLPADDING="4" CELLSPACING="3"> | ||
+ | |||
+ | <TR> | ||
+ | <TH COLSPAN="2"><BR><H4>Ligation</H4></TH> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>Backbone</b></TH> | ||
+ | <TD>2μl</TD> | ||
+ | |||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>Upstream</b></TH> | ||
+ | <TD>2μl</TD> | ||
+ | |||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>Downstream</b></TH> | ||
+ | <TD>2μl</TD> | ||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>DNA ligase buffer</b></TH> | ||
+ | <TD>1μl</TD> | ||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>DNA ligase enzyme</b></TH> | ||
+ | <TD>0.5μL</TD> | ||
+ | |||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>Water</b></TH> | ||
+ | <TD>2.5μL</TD> | ||
+ | </TR> | ||
+ | <TR> | ||
+ | |||
+ | |||
+ | <p>Plasmid + Upstream + Downstream</p> | ||
+ | <p>Plasmid + Tetr_RBS + Cyc_Term</p> | ||
+ | <p>Plasmid + Tetr_RBS + HAS_Term</p> | ||
+ | <p>Plasmid + OmpA + SacB_Term</p> | ||
+ | |||
+ | <p><b>2.50pm</b></p> | ||
+ | <p>Transformations</p> | ||
+ | Our own competent cells were removed from freezer, -80°C. | ||
+ | |||
+ | <ul><b><i>1.</i></b> 5μl of DNA from each sample, Tetr_RBS + Cyc _Term & Has_Term and OmpA + SacB_Term added to 50μl of competent cells. The reason for using the entire quantity of competent cells was because they were believed to less efficient than TOP10. </ul> | ||
+ | <ul><b><i>2.</i></b> Kept on ice for 30 minutes. Whilst heat bath equilibrates to 42°C.</ul> | ||
+ | <p><b>3.20pm</p></b> | ||
+ | <ul><b><i>3.</i></b> Heat shocked cells – transferred to water bath 42°C for 30 seconds exactly. </ul> | ||
+ | <ul><b><i>4.</i></b> Moved straight back into ice for ~2 minutes. </ul> | ||
+ | <ul><b><i>5.</i></b> Aseptically added 700μl of room temperature LG medium to each eppendorf. </ul> | ||
+ | <p><b>3.30pm</p></b> | ||
+ | <ul><b><i>6.</i></b> Incubated all samples in the shaker at 37°C, 250 rpm for 1 hour. </ul></p> | ||
+ | <ul><b><i>7.</i></b> Span samples at 3000G for 5 minutes.<ul> | ||
+ | <p>Plates labelled:</p> | ||
+ | <p>Tetr_RBS + cyc_Term </p> | ||
+ | <p>Tetr_RBS + HAS_Term </p> | ||
+ | <p>OmpA + SacB_Term </p> | ||
+ | <p> Aseptically discarded 600μl solution from each eppendorf. Pipette mixed remaining and spread over plates.</p> | ||
+ | <p><b>5.05pm</p></b> | ||
+ | <p>Plates in incubator, 37°C overnight. </p> | ||
+ | <br> | ||
+ | |||
+ | <p>**<b>Tuesday 04/09/12</b>**</p><br> | ||
+ | |||
+ | <p><b>9.00am</b></p> | ||
+ | <p>Checked plates. Colonies have formed on all. Our own competent cells appear to be more efficient than first expected, less should be spread on future plates to make single colonies easier to pick.</p> | ||
+ | <p>These were all placed in the fridge, 4°C.</p> | ||
+ | |||
+ | <p><b>3.25pm</b></p> | ||
+ | Plates out of fridge, read to make liquid broth. | ||
+ | |||
+ | <p>4x made per plate – aseptic technique</p> | ||
+ | <p> Used 10ml broth</p> | ||
+ | <p> 10μl Kanamycin</p> | ||
+ | <p>Scraped off single colony using pipette tip which was then ejected into liquid broth.</p> | ||
+ | <p>Tetr_RBS + HAS_Term and OmpA + SacB_Term had red and white colonies on the plates. Only the white colonies were selected. | ||
+ | |||
+ | <p><b>4.20pm</b></p> | ||
+ | <p>Put into horizontal shaker set at 37°C, 220rpm – left overnight. | ||
+ | <br><br> | ||
+ | |||
+ | <p>**<b>Wednesday 05/09/12</b>**</p><br> | ||
+ | |||
+ | <p><b>9.00am</b></p> | ||
+ | <p>Liquid broth aseptically transferred to new falcon tubes – leaving pipette tip behind.</p> | ||
+ | <p><b>10.00am</b></p> | ||
+ | <p>These were then centrifuged at 3900rpg for 10 minutes, 4°C.</p> | ||
+ | <p>The same protocol was followed as in the previous week for mini preps. However 40μl of clean water was added, instead of the previous 50μl, to boost the concentration of DNA in each sample. </p> | ||
+ | <p><b>1.30pm</b></p> | ||
+ | |||
+ | <TABLE BORDER="3" ALIGN="CENTER" WIDTH="50%" CELLPADDING="4" CELLSPACING="3"> | ||
+ | |||
+ | <TR> | ||
+ | <TH>Sample</TH> | ||
+ | <TH>Conc of DNA </TH> | ||
+ | <TH>DNA required</TH> | ||
+ | <TH>Water required</TH> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TD><b>Tetr_RBS + Cyc_Term</b></TD> | ||
+ | <TD>ng/μl </TD> | ||
+ | <TD>μl </TD> | ||
+ | <TD>μl </TD> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TD><b>1</b></TD> | ||
+ | <TD>136.8</TD> | ||
+ | <TD>3.65</TD> | ||
+ | <TD>13.15</TD> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TD><b>2</b></TD> | ||
+ | <TD>154.4</TD> | ||
+ | <TD>3.24</TD> | ||
+ | <TD>13.56</TD> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TD><b>3</b></TD> | ||
+ | <TD>126.5 </TD> | ||
+ | <TD> 3.95</TD> | ||
+ | <TD> 12.85</TD> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TD><b>4</b></TD> | ||
+ | <TD>114.8</TD> | ||
+ | <TD>4.36</TD> | ||
+ | <TD>12.44</TD> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TD><b>Tetr_RBS + HAS_Term</b></TD> | ||
+ | <TD>ng/μl </TD> | ||
+ | <TD>μl </TD> | ||
+ | <TD>μl </TD> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TD><b>1</b></TD> | ||
+ | <TD>68.4</TD> | ||
+ | <TD>7.31</TD> | ||
+ | <TD>9.49</TD> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TD><b>2</b></TD> | ||
+ | <TD>65.3</TD> | ||
+ | <TD>7.66</TD> | ||
+ | <TD>9.14</TD> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TD><b>3</b></TD> | ||
+ | <TD>100.6 </TD> | ||
+ | <TD> 4.97</TD> | ||
+ | <TD> 11.83</TD> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TD><b>4</b></TD> | ||
+ | <TD>92.8</TD> | ||
+ | <TD>5.39</TD> | ||
+ | <TD>11.41</TD> | ||
+ | </TR> | ||
+ | |||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TD><b>OmpA + SacB_Term </b></TD> | ||
+ | <TD>ng/μl </TD> | ||
+ | <TD>μl </TD> | ||
+ | <TD>μl </TD> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TD><b>1</b></TD> | ||
+ | <TD>123.6</TD> | ||
+ | <TD>4.05</TD> | ||
+ | <TD>12.75</TD> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TD><b>2</b></TD> | ||
+ | <TD>116.7</TD> | ||
+ | <TD>4.28</TD> | ||
+ | <TD>12.52</TD> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TD><b>3</b></TD> | ||
+ | <TD>250.1 </TD> | ||
+ | <TD> 2.00</TD> | ||
+ | <TD> 14.8</TD> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TD><b>4</b></TD> | ||
+ | <TD>95.7</TD> | ||
+ | <TD>5.22</TD> | ||
+ | <TD>11.58</TD> | ||
+ | </TR> | ||
+ | |||
+ | </TABLE> | ||
+ | <p><b>2.45pm</b></p> | ||
+ | <p>Currently no enzymes, all DNA into freezer. | ||
+ | <p><b>4.30pm</b></p> | ||
+ | <p>Streak plates made from the single colonies that appeared to have worked. Colonies that form should therefore also work.</p> | ||
+ | <ul>HAS_Term 1 & 4 </ul> | ||
+ | <ul>Cyc_Term 1, 2 & 4 </ul> | ||
+ | <ul>SacB_Term 1, 3 & 4 </ul> | ||
+ | <ul>HAS 1, 2, 3 & 4 </ul> | ||
+ | <ul>Cyc 1 & 2 </ul> | ||
+ | <p>All into incubator at 37°C overnight. </p> | ||
+ | |||
+ | <br><br> | ||
+ | |||
+ | <p>**<b>Thursday 06/09/12</b>**</p><br> | ||
+ | |||
+ | <p><b>3.30pm</b></p> | ||
+ | <p> Digestion</p> | ||
+ | |||
+ | |||
+ | <TABLE BORDER="3" ALIGN="CENTRE" WIDTH="50%" CELLPADDING="4" CELLSPACING="3"> | ||
+ | |||
+ | <TR> | ||
+ | <TH COLSPAN="2"><BR><H4>Digestion</H4></TH> | ||
+ | </TR> | ||
+ | |||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>DNA</b></TH> | ||
+ | <TD>500ng</TD> | ||
+ | |||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>BUFFER (10x)</b></TH> | ||
+ | <TD>2μl</TD> | ||
+ | |||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>BSA (100x)</b></TH> | ||
+ | <TD>0.2μl</TD> | ||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>Water</b></TH> | ||
+ | <TD>up to 20μl total V</TD> | ||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>ENZYME 1 - ECORI</b></TH> | ||
+ | <TD>0.5μL</TD> | ||
+ | |||
+ | |||
+ | |||
+ | </TR> | ||
+ | <TR ALIGN="CENTER"> | ||
+ | <TH><b>ENZYME 2 - PstI</b></TH> | ||
+ | <TD>0.5μL</TD> | ||
+ | </TR> | ||
+ | </TABLE> | ||
+ | |||
+ | <p>Into incubator for overnight digestion.</p> | ||
+ | <br> <br> | ||
+ | |||
+ | <p>**<b>Friday 07/09/12</b>**</p><br> | ||
+ | <p><b>11.00am</b></p> | ||
+ | <p>Gel electrophoresis run</p> | ||
+ | |||
+ | |||
+ | <p>Sequences confirmed for:</p> | ||
+ | <ul>Cyclodextrin_Terminator</ul> | ||
+ | <ul>HAS_Terminator</ul> | ||
+ | <ul>SacB_Terminator</ul> | ||
+ | <p>Single genes confirmed for:</p> | ||
+ | <ul>Cyclodextrin</ul> | ||
+ | <ul>HAS</ul> | ||
+ | |||
+ | |||
+ | |||
</font> | </font> | ||
</div> | </div> |
Revision as of 10:27, 13 September 2012
Showcasing Polysaccharide Production: 3rd - 7th September 2012 **Monday 03/09/12** 9.00am Transformations using Gene_Terminator.
Plasmid + Upstream + Downstream Plasmid + Tetr_RBS + Cyc_Term Plasmid + Tetr_RBS + HAS_Term Plasmid + OmpA + SacB_Term 11.20am All in the incubator for digestion period. 12.40pm Heat denatured enzymes at 80°C for 20 minutes. 1.00pm Ligation process
2.45pm Currently no enzymes, all DNA into freezer. 4.30pm Streak plates made from the single colonies that appeared to have worked. Colonies that form should therefore also work.
All into incubator at 37°C overnight. **Thursday 06/09/12** 3.30pm Digestion
Into incubator for overnight digestion. **Friday 07/09/12** 11.00am Gel electrophoresis run Sequences confirmed for:
Single genes confirmed for:
|