http://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Notebook/Methods&feed=atom&action=historyTeam:Heidelberg/pages/Notebook/Methods - Revision history2024-03-28T21:16:13ZRevision history for this page on the wikiMediaWiki 1.16.5http://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Notebook/Methods&diff=403742&oldid=prevBunnech at 16:54, 8 December 20142014-12-08T16:54:40Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Dilute the plasmid DNA.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Dilute the plasmid DNA.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>== Golden Gate Assembly</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>== Golden Gate Assembly<ins class="diffchange diffchange-inline">==</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Mix 150 ng of the backbone and equimolar amounts of the insert(s) in water in a PCR tube for a total volume of 15 µl.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Mix 150 ng of the backbone and equimolar amounts of the insert(s) in water in a PCR tube for a total volume of 15 µl.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Add 1.5 µl of 10X T4 Ligase Buffer and, when using BsaI or another restriction enzyme that requires it, BSA (Bovine Serum Albumin) at a final concentration of 1X.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Add 1.5 µl of 10X T4 Ligase Buffer and, when using BsaI or another restriction enzyme that requires it, BSA (Bovine Serum Albumin) at a final concentration of 1X.</div></td></tr>
</table>Bunnechhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Notebook/Methods&diff=392757&oldid=prevMaxW at 02:57, 18 October 20142014-10-18T02:57:19Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Dilute the plasmid DNA.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Dilute the plasmid DNA.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>== Golden Gate Assembly <del class="diffchange diffchange-inline">(isothermal) ==</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>== Golden Gate Assembly</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># Mix <del class="diffchange diffchange-inline">100 </del>ng of the backbone and equimolar amounts of the insert(s) in water for a total volume of 15 µl.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Mix <ins class="diffchange diffchange-inline">150 </ins>ng of the backbone and equimolar amounts of the insert(s) in water <ins class="diffchange diffchange-inline">in a PCR tube </ins>for a total volume of 15 µl.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Add 1.5 µl of 10X T4 Ligase Buffer and, when using BsaI or another restriction enzyme that requires it, BSA (Bovine Serum Albumin) at a final concentration of 1X.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Add 1.5 µl of 10X T4 Ligase Buffer and, when using BsaI or another restriction enzyme that requires it, BSA (Bovine Serum Albumin) at a final concentration of 1X.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># Add 1 µl of each restriction enzyme and 1 µl of T4 DNA Ligase (<del class="diffchange diffchange-inline">2,000</del>,000 cohesive end ligation units/ml).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Add 1 µl of each restriction enzyme and 1 µl of T4 DNA Ligase (<ins class="diffchange diffchange-inline">400</ins>,000 cohesive end ligation units/ml). </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># <del class="diffchange diffchange-inline">Incubate </del>at <del class="diffchange diffchange-inline">37 </del>°C <del class="diffchange diffchange-inline">for 1 h</del>, <del class="diffchange diffchange-inline">then heat inactivate </del>at 50 °C and 80 °C for 5 min <del class="diffchange diffchange-inline">each</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># <ins class="diffchange diffchange-inline">Optional: Add 1 µl of T4 Polynucleotide Kinase, if several inserts without 5’-phosphorylation (like annealed oligos) are used. </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"># Place the reaction in a thermocycler and run the following program: 25 cycles of 4 min ligation </ins>at <ins class="diffchange diffchange-inline">16 </ins>°C <ins class="diffchange diffchange-inline">and 3 min restriction at 37°C</ins>, <ins class="diffchange diffchange-inline">followed by 5 min </ins>at 50 °C <ins class="diffchange diffchange-inline">for final restriction </ins>and <ins class="diffchange diffchange-inline">5 min at </ins>80 °C for <ins class="diffchange diffchange-inline">heat inactivation. </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">== Religation after Golden gate Assembly ==</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"># Important: This will have a drastic effect on the efficiency, since the original backbone will be able to reassemble. If you don’t use a selection marker, a crazy colony PCR screening will be necessary to find the desired plasmid. </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"># Add 12.</ins>5 <ins class="diffchange diffchange-inline">µl of water, 1.5 µl of T4 Ligase Buffer and 1 µl of T4 DNA Ligase to your Golden Gate reaction. </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"># Incubate for 20 </ins>min <ins class="diffchange diffchange-inline">at 16 °C, then heat inactivate for 10 min at 65 °C</ins>. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Make competent cells already carrying a plasmid ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Make competent cells already carrying a plasmid ==</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Freeze at -80 °C.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Freeze at -80 °C.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>== CPEC ==</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>==CPEC==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># <del class="diffchange diffchange-inline">Mix </del>the <del class="diffchange diffchange-inline">DNA </del>fragments at the <del class="diffchange diffchange-inline">desired ratio</del>. <del class="diffchange diffchange-inline">Optionally add </del>a <del class="diffchange diffchange-inline">small amount </del>of <del class="diffchange diffchange-inline">DMSO</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># <ins class="diffchange diffchange-inline">Design of the insert and backbone primers</ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># Add 2X Phusion Flash Master Mix</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">**Assemble your desired construct in silico by pasting </ins>the fragments<ins class="diffchange diffchange-inline">. Feel free to add tags and other small stuff between them. </ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"># Place the reaction in </del>a thermocycler <del class="diffchange diffchange-inline">and run the following program</del>: Initial denaturation <del class="diffchange diffchange-inline">at </del>98 °C <del class="diffchange diffchange-inline">for </del>10 s<del class="diffchange diffchange-inline">; </del>1 <del class="diffchange diffchange-inline">to </del>30 cycles <del class="diffchange diffchange-inline">of </del>1 s denaturation <del class="diffchange diffchange-inline">at </del>98 °C, 5 s <del class="diffchange diffchange-inline">annealing, extension at </del>72 °C <del class="diffchange diffchange-inline">for </del>15 <del class="diffchange diffchange-inline">to 30 </del>s/kb <del class="diffchange diffchange-inline">of the longest fragment; final extension at </del>72 °C <del class="diffchange diffchange-inline">for </del>5 <del class="diffchange diffchange-inline">min.</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">**Define a “homology region” with a Tm of 60 °C - 70 °C (~15 - 35 bp) </ins>at <ins class="diffchange diffchange-inline">each fragment border. The Tms of all homology regions should be very similar. It is your choice whether </ins>the <ins class="diffchange diffchange-inline">regions are right next to the border in one fragment, include parts of both fragments, include inserted sequences</ins>. <ins class="diffchange diffchange-inline">These regions should be checked carefully for annoying secondary structures. </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">**You need </ins>a <ins class="diffchange diffchange-inline">forward and a reverse primer for any fragment. Any primer consists </ins>of <ins class="diffchange diffchange-inline">a part that binds to the template DNA and an overlap</ins>. <ins class="diffchange diffchange-inline">The binding part should have a G or C at the 3’ end and a Tm between 60 °C and 72 °C. The binding part Tms of one primer pair should be similar. In addition to the binding part, any primer must have an overlap part. Just extend the primers until the chosen homology region is fully covered. Check the complete primers for secondary structures. If there are really bad ones, you should change the homology region…</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># <ins class="diffchange diffchange-inline">PCRs of the inserts</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">**Please follow the [[#Touchdown-Twostep PCR (Phusion Flash)|Touchdown-Twostep PCR protocol]], purify the products and measure their concentration. </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">#The CPEC</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">**Mix your fragments. At least 100 ng vector are required, we recommend to use around 500 ng/µl. </ins>Add <ins class="diffchange diffchange-inline">equimolar amounts of your insert(s). A vector: insert ratio of 1:3 works as well. </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">**Add 10 µl </ins>2X Phusion Flash Master Mix<ins class="diffchange diffchange-inline">.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">**Add H2O to </ins>a <ins class="diffchange diffchange-inline">final volume of 20 µl. </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">**Run your </ins>thermocycler: </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">{| class="table table-hover”</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">|-</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">! Step !! Temperature !! Time</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">|-</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">| </ins>Initial denaturation <ins class="diffchange diffchange-inline"> || </ins>98 °C <ins class="diffchange diffchange-inline"> || </ins>10 s</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">|-</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">| </ins>1 <ins class="diffchange diffchange-inline">insert: 10 cycles, >1 insert: 20-</ins>30 cycles <ins class="diffchange diffchange-inline"> || ||</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">|-</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">| - Denaturation || 98 °C || </ins>1 s</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">|-</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">| - Annealing || Slow ramping from 70°C to 55°C (-0.1 °C/ s) || ~3 min</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">|-</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">| - Elongation || 72 °C || 15 s / kb (desired plasmid)</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">|-</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">| End elongation || 72 °C || 120 s</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">|}</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">==Touchdown-Twostep PCR (Phusion Flash)==</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"># Add H2O to a final volume of 50 µl.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"># Add 25 µl 2X Phusion Flash Master Mix.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"># Add primers to a final concentration of 0.5 µM.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"># Add 2.5 pg to 25 ng template DNA.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"># Run your thermocycler: </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">{| class="table table-hover”</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">|-</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">! Step !! Temperature !! Time</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">|-</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">| Initial </ins>denaturation <ins class="diffchange diffchange-inline"> || </ins>98 <ins class="diffchange diffchange-inline">°C || 10 s</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">|-</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">| Touchdown (~12 cycles) || ||</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">|-</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">| - Denaturation || 98 °C || 1 s</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">|-</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">| - Annealing || Primer Tm + 4 </ins>°C, <ins class="diffchange diffchange-inline">-0.5 °C each cycle || </ins>5 s</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">|-</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">| - Elongation || </ins>72 °C <ins class="diffchange diffchange-inline">|| </ins>15 s / kb</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">|-</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">| Twostep (~24 cycles) || || </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">|-</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">| - Denaturation || 98 °C || 1 s</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">|-</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">| - Elongation || </ins>72 °C <ins class="diffchange diffchange-inline">|| </ins>5 <ins class="diffchange diffchange-inline">s + 15 s /kb</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">|-</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">| End elongation || 72 °C || 120 s</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">|}</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Lysozyme Assay ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Lysozyme Assay ==</div></td></tr>
</table>MaxWhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Notebook/Methods&diff=376966&oldid=prevJakob: /* CPEC */2014-10-18T00:43:28Z<p><span class="autocomment">CPEC</span></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 00:43, 18 October 2014</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Add 2X Phusion Flash Master Mix</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Add 2X Phusion Flash Master Mix</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Place the reaction in a thermocycler and run the following program: Initial denaturation at 98 °C for 10 s; 1 to 30 cycles of 1 s denaturation at 98 °C, 5 s annealing, extension at 72 °C for 15 to 30 s/kb of the longest fragment; final extension at 72 °C for 5 min.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Place the reaction in a thermocycler and run the following program: Initial denaturation at 98 °C for 10 s; 1 to 30 cycles of 1 s denaturation at 98 °C, 5 s annealing, extension at 72 °C for 15 to 30 s/kb of the longest fragment; final extension at 72 °C for 5 min.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">---------------------------------------------------------------------------- manually added stuff below!</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Lysozyme Assay ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Lysozyme Assay ==</div></td></tr>
</table>Jakobhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Notebook/Methods&diff=288392&oldid=prevBunnech at 20:56, 16 October 20142014-10-16T20:56:18Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Find here a collection of all the methods we used for cloning our constructs and performing our assays. For the individual experiments visit our Notebook, the <a style="font-weight:bold" href="https://2014.igem.org/Team:Heidelberg/Notebook">MidnightDoc</a>, for the Materials we used, visit <a href="https://2014.igem.org/Team:Heidelberg/Notebook/Materials">Materials</a>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><html></ins><p>Find here a collection of all the methods we used for cloning our constructs and performing our assays. For the individual experiments visit our Notebook, the <a style="font-weight:bold" href="https://2014.igem.org/Team:Heidelberg/Notebook">MidnightDoc</a>, for the Materials we used, visit <a href="https://2014.igem.org/Team:Heidelberg/Notebook/Materials">Materials</a>.</p<ins class="diffchange diffchange-inline">></html</ins>></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Make a liquid culture without any plasmid ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Make a liquid culture without any plasmid ==</div></td></tr>
</table>Bunnechhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Notebook/Methods&diff=288379&oldid=prevBunnech at 20:56, 16 October 20142014-10-16T20:56:04Z<p></p>
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<td colspan="2" class="diff-lineno">Line 1:</td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Find here a collection of all the methods we used for cloning our constructs and performing our assays. For the individual experiments visit our Notebook, the <a style="font-weight:bold" href="https://2014.igem.org/Team:Heidelberg/Notebook<del class="diffchange diffchange-inline">/Materials</del>">MidnightDoc</a>, for the Materials we used, visit <a href="https://2014.igem.org/Team:Heidelberg/Notebook/Materials">Materials</a>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Find here a collection of all the methods we used for cloning our constructs and performing our assays. For the individual experiments visit our Notebook, the <a style="font-weight:bold" href="https://2014.igem.org/Team:Heidelberg/Notebook">MidnightDoc</a>, for the Materials we used, visit <a href="https://2014.igem.org/Team:Heidelberg/Notebook/Materials">Materials</a>.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Make a liquid culture without any plasmid ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Make a liquid culture without any plasmid ==</div></td></tr>
</table>Bunnechhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Notebook/Methods&diff=288344&oldid=prevBunnech at 20:55, 16 October 20142014-10-16T20:55:16Z<p></p>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><p>Find here a collection of all the methods we used for cloning our constructs and performing our assays. For the individual experiments visit our Notebook, the <a style="font-weight:bold" href="https://2014.igem.org/Team:Heidelberg/Notebook/Materials">MidnightDoc</a>, for the Materials we used, visit <a href="https://2014.igem.org/Team:Heidelberg/Notebook/Materials">Materials</a>.</p></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Make a liquid culture without any plasmid ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Make a liquid culture without any plasmid ==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Inoculate medium</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Inoculate medium</div></td></tr>
</table>Bunnechhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Notebook/Methods&diff=280019&oldid=prevJakob at 16:46, 16 October 20142014-10-16T16:46:57Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Make a liquid culture without any plasmid ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Make a liquid culture without any plasmid ==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Inoculate medium</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Inoculate medium</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Add 2X Phusion Flash Master Mix</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Add 2X Phusion Flash Master Mix</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Place the reaction in a thermocycler and run the following program: Initial denaturation at 98 °C for 10 s; 1 to 30 cycles of 1 s denaturation at 98 °C, 5 s annealing, extension at 72 °C for 15 to 30 s/kb of the longest fragment; final extension at 72 °C for 5 min.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Place the reaction in a thermocycler and run the following program: Initial denaturation at 98 °C for 10 s; 1 to 30 cycles of 1 s denaturation at 98 °C, 5 s annealing, extension at 72 °C for 15 to 30 s/kb of the longest fragment; final extension at 72 °C for 5 min.</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">---------------------------------------------------------------------------- manually added stuff below!</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Lysozyme Assay ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Lysozyme Assay ==</div></td></tr>
</table>Jakobhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Notebook/Methods&diff=277048&oldid=prevJakob at 15:26, 16 October 20142014-10-16T15:26:12Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Make a liquid culture without any plasmid ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Make a liquid culture without any plasmid ==</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Add 2X Phusion Flash Master Mix</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Add 2X Phusion Flash Master Mix</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Place the reaction in a thermocycler and run the following program: Initial denaturation at 98 °C for 10 s; 1 to 30 cycles of 1 s denaturation at 98 °C, 5 s annealing, extension at 72 °C for 15 to 30 s/kb of the longest fragment; final extension at 72 °C for 5 min.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Place the reaction in a thermocycler and run the following program: Initial denaturation at 98 °C for 10 s; 1 to 30 cycles of 1 s denaturation at 98 °C, 5 s annealing, extension at 72 °C for 15 to 30 s/kb of the longest fragment; final extension at 72 °C for 5 min.</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">== Lysozyme Assay ==</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Bacteria were transformed with lambda lysozyme constructs, grown to an OD of 0.6 and induced with 1 mM IPTG.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">After 4 hours of expression, the samples were diluted to the same OD of 2.0 and centrifuged down at 2,850 rpm. The pellet was resuspended in 10 mM potassium phosphate buffer with a pH of 6.24 and sonicated for two minutes on ice. After centrifugation a second time at 2,850 rpm, the supernatant containing the lambda lysozyme was kept.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">For substrate preparation, the lyophilized cells of M.lysodeikticus (Sigma Aldrich), were resuspended in ultrapure water. The supernantant with the protein mix was transfered and aliquoted for biological replicates and to prepare dilution series.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Subsequently the samples were transfered into the thermocycler to do a one minute heat-shock in a temperature span between 45 and 55°C. After mixing enzyme and substrate the OD was measured every two minutes in a plate reader over 100 minutes at 37°C (figure 4).</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">=== Flourescence labeled peptidoglycane assay</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">The expression of the lysozyme samples for the assay using FITC-labeled peptidoglycan was carried out in the same way as we did it in the first assay. We resuspended the pellet in PBS. The protocol of labeling of peptidoglycan with fluorescein isothiocyanate (FITC) you can find in this methods section as well.</ins></div></td></tr>
</table>Jakobhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Notebook/Methods&diff=224574&oldid=prevJakob at 09:59, 14 October 20142014-10-14T09:59:02Z<p></p>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Make a liquid culture without any plasmid ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Make a liquid culture without any plasmid ==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Inoculate medium</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Inoculate medium</div></td></tr>
</table>Jakobhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Notebook/Methods&diff=224533&oldid=prevJakob at 09:56, 14 October 20142014-10-14T09:56:03Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">_NOTOC_</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Make a liquid culture without any plasmid ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Make a liquid culture without any plasmid ==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Inoculate medium</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Inoculate medium</div></td></tr>
</table>Jakob