http://2009.igem.org/wiki/index.php?title=Team:Victoria_Australia/Project/Materials_and_Methods&feed=atom&action=historyTeam:Victoria Australia/Project/Materials and Methods - Revision history2024-03-29T13:21:42ZRevision history for this page on the wikiMediaWiki 1.16.5http://2009.igem.org/wiki/index.php?title=Team:Victoria_Australia/Project/Materials_and_Methods&diff=139256&oldid=prevDannii: /* Mutagenesis of YFP */2009-10-21T06:43:13Z<p><span class="autocomment">Mutagenesis of YFP</span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 06:43, 21 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 173:</td>
<td colspan="2" class="diff-lineno">Line 173:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|- </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|- </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-valign="top" </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-valign="top" </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>|width="50%"|1µL of the Dpn I restriction enyme at 10 U/µL was directly added the <del class="diffchange diffchange-inline">amplifictaion </del>reaction in the tube and using a pipette it was <del class="diffchange diffchange-inline">thoroguhly </del>mixed by pipetting the solution up and down several times. The reaction mixture was <del class="diffchange diffchange-inline">microcentrifuged</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|width="50%"|1µL of the Dpn I restriction enyme at 10 U/µL was directly added the <ins class="diffchange diffchange-inline">amplification </ins>reaction in the tube and using a pipette it was <ins class="diffchange diffchange-inline">thoroughly </ins>mixed by pipetting the solution up and down several times. The reaction mixture was <ins class="diffchange diffchange-inline">micro centrifuged </ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%" | for 1 minute and incubated at 37°C for 7 hours to digest the parental supercoiled dsDNA.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%" | for 1 minute and incubated at 37°C for 7 hours to digest the parental supercoiled dsDNA.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 184:</td>
<td colspan="2" class="diff-lineno">Line 184:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-valign="top" </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-valign="top" </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%"|The XL-10 Gold ultracompetent cells were thawed on ice before aliquoting 45µL into 4 eppendorf tube. 2µL of the Beta Mecaptoethanol was added to the cells and swirled gently. 2µLof the Dpn I treated DNA was added to the aliquotes of the ultracompetent cells to seperate them. It was then incubated on ice for 30 minutes. After the incubation period, the cells were heat shocked by transferring</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%"|The XL-10 Gold ultracompetent cells were thawed on ice before aliquoting 45µL into 4 eppendorf tube. 2µL of the Beta Mecaptoethanol was added to the cells and swirled gently. 2µLof the Dpn I treated DNA was added to the aliquotes of the ultracompetent cells to seperate them. It was then incubated on ice for 30 minutes. After the incubation period, the cells were heat shocked by transferring</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>|width="50%"|the tubes in a 42°C water bath for 90 seconds and then <del class="diffchange diffchange-inline">immediatly </del>placing the tubes on ice for 2 minutes. 800µL of SOC medium was added to each tube and incubated for 45 minutes at 37°C and 225rpm. Following incubation, samples from each tube being 50µL, 100µL, 150µL and 200µL were added to a LB agar plate with ampicillin and again incubated at 37°C overnight.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|width="50%"|the tubes in a 42°C water bath for 90 seconds and then <ins class="diffchange diffchange-inline">immediately </ins>placing the tubes on ice for 2 minutes. 800µL of SOC medium was added to each tube and incubated for 45 minutes at 37°C and 225rpm. Following incubation, samples from each tube being 50µL, 100µL, 150µL and 200µL were added to a LB agar plate with ampicillin and again incubated at 37°C overnight.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td></tr>
</table>Danniihttp://2009.igem.org/wiki/index.php?title=Team:Victoria_Australia/Project/Materials_and_Methods&diff=137198&oldid=prevDannii: /* Purification of GFP and BFP */2009-10-21T02:42:24Z<p><span class="autocomment">Purification of GFP and BFP</span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 02:42, 21 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 45:</td>
<td colspan="2" class="diff-lineno">Line 45:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-valign="top" </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-valign="top" </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%"|1ml lysis buffer was then added to pellets containing either GFP/BFP in order to resuspend the pellet. After resuspending, a Pasteur pipette was used to remove 1ml into another pellet also containing GFP or BFP. These were sonicated to split the cells open at 70% duty cycle 30seconds on and 20seconds off for 3 cycles. Samples then centrifuged for 40min. The supernatant was transferred into eppendorf tubes (2 times GFP and 2times BFP). 250 µL of resin was added to each tube and placed on suspension mixer for 20min. again, the supernatant was removed and added 1ml phosphate buffer to the remaining resin. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%"|1ml lysis buffer was then added to pellets containing either GFP/BFP in order to resuspend the pellet. After resuspending, a Pasteur pipette was used to remove 1ml into another pellet also containing GFP or BFP. These were sonicated to split the cells open at 70% duty cycle 30seconds on and 20seconds off for 3 cycles. Samples then centrifuged for 40min. The supernatant was transferred into eppendorf tubes (2 times GFP and 2times BFP). 250 µL of resin was added to each tube and placed on suspension mixer for 20min. again, the supernatant was removed and added 1ml phosphate buffer to the remaining resin. </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>This was centrifuged at 500rpm for 2min and the supernatant removed with 1ml phosphate buffer being again added. This was centrifuged at 500rpm for 2min and the supernatant removed. The pellet was resuspended with 5ml elution buffer (An elution buffer was made using 2.5ml <del class="diffchange diffchange-inline">imieazole </del>with phosphate buffer being added resulting in a total volume of 10ml. ) again this was centrifuged for 5min at 500rpm. The resulting supernatant formed contained the GFP and BFP; this was removed. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>This was centrifuged at 500rpm for 2min and the supernatant removed with 1ml phosphate buffer being again added. This was centrifuged at 500rpm for 2min and the supernatant removed. The pellet was resuspended with 5ml elution buffer (An elution buffer was made using 2.5ml <ins class="diffchange diffchange-inline">imidazole </ins>with phosphate buffer being added resulting in a total volume of 10ml. ) again this was centrifuged for 5min at 500rpm. The resulting supernatant formed contained the GFP and BFP; this was removed. </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;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
</table>Danniihttp://2009.igem.org/wiki/index.php?title=Team:Victoria_Australia/Project/Materials_and_Methods&diff=137187&oldid=prevDannii: /* Preparation of reagents */2009-10-21T02:41:44Z<p><span class="autocomment">Preparation of reagents</span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 02:41, 21 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 37:</td>
<td colspan="2" class="diff-lineno">Line 37:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-valign="top" </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-valign="top" </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%"|Following these transformations, more reagents were prepared. Phosphate buffer solution was made by adding 1 tablet of phosphate buffered saline to 100ml milliQ water. Chaps had also been made using 0.017g chaps in 1ml of milliQ water. Lysis Buffer 1 was created using 250 µL chaps, 500 µL sucrose and made up to 10ml using the phosphate buffer. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%"|Following these transformations, more reagents were prepared. Phosphate buffer solution was made by adding 1 tablet of phosphate buffered saline to 100ml milliQ water. Chaps had also been made using 0.017g chaps in 1ml of milliQ water. Lysis Buffer 1 was created using 250 µL chaps, 500 µL sucrose and made up to 10ml using the phosphate buffer. </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>|width="50%" |100ml milliQ water was added to 34.2g sucrose making 1M sucrose solution. 1M imidazole was prepared by adding 0.6808g of <del class="diffchange diffchange-inline">imieazole </del>to 10ml of milliQ water. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|width="50%" |100ml milliQ water was added to 34.2g sucrose making 1M sucrose solution. 1M imidazole was prepared by adding 0.6808g of <ins class="diffchange diffchange-inline">imidazole </ins>to 10ml of milliQ water. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'> </td><td style="background: #eee; 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>
</table>Danniihttp://2009.igem.org/wiki/index.php?title=Team:Victoria_Australia/Project/Materials_and_Methods&diff=137177&oldid=prevDannii: /* Preparation of reagents */2009-10-21T02:41:19Z<p><span class="autocomment">Preparation of reagents</span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 02:41, 21 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 37:</td>
<td colspan="2" class="diff-lineno">Line 37:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-valign="top" </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-valign="top" </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%"|Following these transformations, more reagents were prepared. Phosphate buffer solution was made by adding 1 tablet of phosphate buffered saline to 100ml milliQ water. Chaps had also been made using 0.017g chaps in 1ml of milliQ water. Lysis Buffer 1 was created using 250 µL chaps, 500 µL sucrose and made up to 10ml using the phosphate buffer. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%"|Following these transformations, more reagents were prepared. Phosphate buffer solution was made by adding 1 tablet of phosphate buffered saline to 100ml milliQ water. Chaps had also been made using 0.017g chaps in 1ml of milliQ water. Lysis Buffer 1 was created using 250 µL chaps, 500 µL sucrose and made up to 10ml using the phosphate buffer. </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>|width="50%" |100ml milliQ water was added to 34.2g sucrose making 1M sucrose solution. 1M <del class="diffchange diffchange-inline">imieazole </del>was prepared by adding 0.6808g of imieazole to 10ml of milliQ water. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|width="50%" |100ml milliQ water was added to 34.2g sucrose making 1M sucrose solution. 1M <ins class="diffchange diffchange-inline">imidazole </ins>was prepared by adding 0.6808g of imieazole to 10ml of milliQ water. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'> </td><td style="background: #eee; 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 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>==Purification of GFP and BFP==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Purification of GFP and BFP==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| style="text-align:center"; width="100%"</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| style="text-align:center"; width="100%"</div></td></tr>
</table>Danniihttp://2009.igem.org/wiki/index.php?title=Team:Victoria_Australia/Project/Materials_and_Methods&diff=136738&oldid=prevDannii: /* Purification of GFP and BFP */2009-10-21T02:06:03Z<p><span class="autocomment">Purification of GFP and BFP</span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 02:06, 21 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 43:</td>
<td colspan="2" class="diff-lineno">Line 43:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|- </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|- </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-valign="top" </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-valign="top" </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>|width="50%"|1ml lysis buffer was then added to pellets containing either GFP/BFP in order to resuspend the pellet. After resuspending, a Pasteur pipette was used to remove 1ml into another pellet also containing GFP or <del class="diffchange diffchange-inline">BFP. That is; GFP with lysis buffer added to pellet with GFP and BFP with lysis buffer added to pellet with </del>BFP. These were sonicated to split the cells open at 70% duty cycle 30seconds on and 20seconds off for 3 cycles. Samples then centrifuged for 40min. The supernatant was transferred into eppendorf tubes (2 times GFP and 2times BFP). 250 µL of resin was added to each tube and placed on suspension mixer for 20min. again, the supernatant was removed and added 1ml phosphate buffer to the remaining resin. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|width="50%"|1ml lysis buffer was then added to pellets containing either GFP/BFP in order to resuspend the pellet. After resuspending, a Pasteur pipette was used to remove 1ml into another pellet also containing GFP or BFP. These were sonicated to split the cells open at 70% duty cycle 30seconds on and 20seconds off for 3 cycles. Samples then centrifuged for 40min. The supernatant was transferred into eppendorf tubes (2 times GFP and 2times BFP). 250 µL of resin was added to each tube and placed on suspension mixer for 20min. again, the supernatant was removed and added 1ml phosphate buffer to the remaining resin. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This was centrifuged at 500rpm for 2min and the supernatant removed with 1ml phosphate buffer being again added. This was centrifuged at 500rpm for 2min and the supernatant removed. The pellet was resuspended with 5ml elution buffer (An elution buffer was made using 2.5ml imieazole with phosphate buffer being added resulting in a total volume of 10ml. ) again this was centrifuged for 5min at 500rpm. The resulting supernatant formed contained the GFP and BFP; this was removed. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This was centrifuged at 500rpm for 2min and the supernatant removed with 1ml phosphate buffer being again added. This was centrifuged at 500rpm for 2min and the supernatant removed. The pellet was resuspended with 5ml elution buffer (An elution buffer was made using 2.5ml imieazole with phosphate buffer being added resulting in a total volume of 10ml. ) again this was centrifuged for 5min at 500rpm. The resulting supernatant formed contained the GFP and BFP; this was removed. </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>
</table>Danniihttp://2009.igem.org/wiki/index.php?title=Team:Victoria_Australia/Project/Materials_and_Methods&diff=135482&oldid=prevMicha: /* Electrophoresis of YFP */2009-10-20T23:58:24Z<p><span class="autocomment">Electrophoresis of YFP</span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 23:58, 20 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 101:</td>
<td colspan="2" class="diff-lineno">Line 101:</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;"></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>|width="50%"|[[Image:SNC00109.jpg|300px|centre|<del class="diffchange diffchange-inline">Agarose gel prepared for the electrophoresis </del>of YFP|thumb]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|width="50%"|[[Image:SNC00109.jpg|300px|centre|<ins class="diffchange diffchange-inline">Electrophoresis run </ins>of <ins class="diffchange diffchange-inline">the </ins>YFP <ins class="diffchange diffchange-inline">plasmid</ins>|thumb]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr>
</table>Michahttp://2009.igem.org/wiki/index.php?title=Team:Victoria_Australia/Project/Materials_and_Methods&diff=135479&oldid=prevMicha: /* Purification of GFP and BFP */2009-10-20T23:57:30Z<p><span class="autocomment">Purification of GFP and BFP</span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 23:57, 20 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 48:</td>
<td colspan="2" class="diff-lineno">Line 48:</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>|width="50%"|</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%"|</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:SNC00184.jpg|300px|centre|Purified GFP and BFP <del class="diffchange diffchange-inline">obtained in the supernatant</del>|thumb]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:SNC00184.jpg|300px|centre|Purified GFP and BFP |thumb]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'> </td><td style="background: #eee; 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>
</table>Michahttp://2009.igem.org/wiki/index.php?title=Team:Victoria_Australia/Project/Materials_and_Methods&diff=135465&oldid=prevMicha at 23:54, 20 October 20092009-10-20T23:54:24Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 23:54, 20 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 174:</td>
<td colspan="2" class="diff-lineno">Line 174:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%"|1µL of the Dpn I restriction enyme at 10 U/µL was directly added the amplifictaion reaction in the tube and using a pipette it was thoroguhly mixed by pipetting the solution up and down several times. The reaction mixture was microcentrifuged</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%"|1µL of the Dpn I restriction enyme at 10 U/µL was directly added the amplifictaion reaction in the tube and using a pipette it was thoroguhly mixed by pipetting the solution up and down several times. The reaction mixture was microcentrifuged</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%" | for 1 minute and incubated at 37°C for 7 hours to digest the parental supercoiled dsDNA.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%" | for 1 minute and incubated at 37°C for 7 hours to digest the parental supercoiled dsDNA.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></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 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;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
</table>Michahttp://2009.igem.org/wiki/index.php?title=Team:Victoria_Australia/Project/Materials_and_Methods&diff=135461&oldid=prevMicha at 23:53, 20 October 20092009-10-20T23:53:29Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 23:53, 20 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 184:</td>
<td colspan="2" class="diff-lineno">Line 184:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%"|The XL-10 Gold ultracompetent cells were thawed on ice before aliquoting 45µL into 4 eppendorf tube. 2µL of the Beta Mecaptoethanol was added to the cells and swirled gently. 2µLof the Dpn I treated DNA was added to the aliquotes of the ultracompetent cells to seperate them. It was then incubated on ice for 30 minutes. After the incubation period, the cells were heat shocked by transferring</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%"|The XL-10 Gold ultracompetent cells were thawed on ice before aliquoting 45µL into 4 eppendorf tube. 2µL of the Beta Mecaptoethanol was added to the cells and swirled gently. 2µLof the Dpn I treated DNA was added to the aliquotes of the ultracompetent cells to seperate them. It was then incubated on ice for 30 minutes. After the incubation period, the cells were heat shocked by transferring</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%"|the tubes in a 42°C water bath for 90 seconds and then immediatly placing the tubes on ice for 2 minutes. 800µL of SOC medium was added to each tube and incubated for 45 minutes at 37°C and 225rpm. Following incubation, samples from each tube being 50µL, 100µL, 150µL and 200µL were added to a LB agar plate with ampicillin and again incubated at 37°C overnight.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%"|the tubes in a 42°C water bath for 90 seconds and then immediatly placing the tubes on ice for 2 minutes. 800µL of SOC medium was added to each tube and incubated for 45 minutes at 37°C and 225rpm. Following incubation, samples from each tube being 50µL, 100µL, 150µL and 200µL were added to a LB agar plate with ampicillin and again incubated at 37°C overnight.</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>
</table>Michahttp://2009.igem.org/wiki/index.php?title=Team:Victoria_Australia/Project/Materials_and_Methods&diff=135447&oldid=prevMicha at 23:51, 20 October 20092009-10-20T23:51:43Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 23:51, 20 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 174:</td>
<td colspan="2" class="diff-lineno">Line 174:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%"|1µL of the Dpn I restriction enyme at 10 U/µL was directly added the amplifictaion reaction in the tube and using a pipette it was thoroguhly mixed by pipetting the solution up and down several times. The reaction mixture was microcentrifuged</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%"|1µL of the Dpn I restriction enyme at 10 U/µL was directly added the amplifictaion reaction in the tube and using a pipette it was thoroguhly mixed by pipetting the solution up and down several times. The reaction mixture was microcentrifuged</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%" | for 1 minute and incubated at 37°C for 7 hours to digest the parental supercoiled dsDNA.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="50%" | for 1 minute and incubated at 37°C for 7 hours to digest the parental supercoiled dsDNA.</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;"></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>'''''Transformation of XL10-Gold Ultracompetent cells'''''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''''Transformation of XL10-Gold Ultracompetent cells'''''</div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 180:</td>
<td colspan="2" class="diff-lineno">Line 182:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|- </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|- </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-valign="top" </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-valign="top" </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>|width="50%"|The XL-10 Gold ultracompetent cells were thawed on ice before aliquoting 45µL into 4 eppendorf tube. 2µL of the Beta Mecaptoethanol was added to the cells and swirled gently. 2µLof the Dpn I treated DNA was added to the aliquotes of the ultracompetent cells to seperate them. It was then incubated on ice for 30 minutes. After the incubation period, the cells were heat shocked by transferring the tubes in a 42°C water bath for 90 seconds and then immediatly placing the tubes on ice for 2 minutes. 800µL of SOC medium was added to each tube and incubated for 45 minutes at 37°C and 225rpm. Following incubation, samples from each tube being 50µL, 100µL, 150µL and 200µL were added to a LB agar plate with ampicillin and again incubated at 37°C overnight.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|width="50%"|The XL-10 Gold ultracompetent cells were thawed on ice before aliquoting 45µL into 4 eppendorf tube. 2µL of the Beta Mecaptoethanol was added to the cells and swirled gently. 2µLof the Dpn I treated DNA was added to the aliquotes of the ultracompetent cells to seperate them. It was then incubated on ice for 30 minutes. After the incubation period, the cells were heat shocked by transferring</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">|width="50%"|</ins>the tubes in a 42°C water bath for 90 seconds and then immediatly placing the tubes on ice for 2 minutes. 800µL of SOC medium was added to each tube and incubated for 45 minutes at 37°C and 225rpm. Following incubation, samples from each tube being 50µL, 100µL, 150µL and 200µL were added to a LB agar plate with ampicillin and again incubated at 37°C overnight.</div></td></tr>
</table>Micha