http://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Material_Methods&feed=atom&action=historyTeam:Heidelberg/Notebook/Material Methods - Revision history2024-03-29T12:01:26ZRevision history for this page on the wikiMediaWiki 1.16.5http://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Material_Methods&diff=205103&oldid=prevOla at 02:14, 28 October 20102010-10-28T02:14:00Z<p></p>
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</table>Olahttp://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Material_Methods&diff=143949&oldid=prevKleinsorg: /* Methods */2010-10-25T12:37:00Z<p><span class="autocomment">Methods</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Cloning==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Cloning==</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>===<del class="diffchange diffchange-inline">Plasmid-DNA isolation</del>===</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>===<ins class="diffchange diffchange-inline">Agarose Gel Electrophoresis</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">5 ml LB</del>-<del class="diffchange diffchange-inline">Medium with 5 µl ampicillin was inoculated with single colonies which grew overnight on a shaker at 37°C</del>. <del class="diffchange diffchange-inline">The plasmid DNA was isolated using QiAprep Spin Miniprep kit from Qiagen </del>and <del class="diffchange diffchange-inline">following the manufacturer’s protocol</del>. <del class="diffchange diffchange-inline">4 ml </del>of <del class="diffchange diffchange-inline">each overnight culture </del>was <del class="diffchange diffchange-inline">pelleted </del>in <del class="diffchange diffchange-inline">2 ml microcentrifuge tubes during two steps of centrifugation at 13</del>.<del class="diffchange diffchange-inline">000 rpm</del>. <del class="diffchange diffchange-inline">Subsequently </del>the <del class="diffchange diffchange-inline">pellet </del>was <del class="diffchange diffchange-inline">resuspended </del>in <del class="diffchange diffchange-inline">250 µl of chilled </del>buffer <del class="diffchange diffchange-inline">P1</del>. <del class="diffchange diffchange-inline">250 µl of lysis </del>buffer <del class="diffchange diffchange-inline">P2 </del>was added <del class="diffchange diffchange-inline">and </del>the <del class="diffchange diffchange-inline">solution </del>was <del class="diffchange diffchange-inline">mixed thoroughly by inverting </del>the <del class="diffchange diffchange-inline">tube 4-6 times</del>. <del class="diffchange diffchange-inline">After adding 350 µl of </del>the <del class="diffchange diffchange-inline">neutralization Buffer N3 the solution was mixed immediately and thoroughly by inverting the tube 4</del>-<del class="diffchange diffchange-inline">6 times</del>. <del class="diffchange diffchange-inline">Thereafter </del>the <del class="diffchange diffchange-inline">mixture was centrifuged. The supernatants were applied to a QIAprep column which was put in </del>a <del class="diffchange diffchange-inline">2 ml collection tube. It was centrifuged for 1 min </del>at <del class="diffchange diffchange-inline">13</del>.<del class="diffchange diffchange-inline">000 rpm and the flow-through was discarded. After adding 500 µl of wash buffer PB</del>, <del class="diffchange diffchange-inline">it was centrifuged for 1 min at 13</del>.<del class="diffchange diffchange-inline">000 rpm </del>and <del class="diffchange diffchange-inline">the flow</del>-<del class="diffchange diffchange-inline">through was discarded. Once more, it was washed </del>with <del class="diffchange diffchange-inline">750 µl of wash buffer PE. In an additional centrifugation for 1 min at 13.000 rpm the residual wash buffer was removed</del>. The <del class="diffchange diffchange-inline">QIAprep column </del>was <del class="diffchange diffchange-inline">placed into a clean 1.5 ml microcentrifuge tube </del>and <del class="diffchange diffchange-inline">the plasmid DNA was eluted in 30 µl ddH2O</del>.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Agarose flat</ins>-<ins class="diffchange diffchange-inline">bed gels in various concentrations (0</ins>.<ins class="diffchange diffchange-inline">6–2% agarose in 0.5 x TAE buffer) </ins>and <ins class="diffchange diffchange-inline">sizes were run to separate DNA fragments in an electrical field (10–20 V/cm) for analytical or preparative use</ins>. <ins class="diffchange diffchange-inline">The desired amount </ins>of <ins class="diffchange diffchange-inline">agarose </ins>was <ins class="diffchange diffchange-inline">boiled </ins>in <ins class="diffchange diffchange-inline">1 x TAE buffer until it was completely dissolved</ins>. <ins class="diffchange diffchange-inline">After it cooled down to approximately 60°C, ethidium bromide (EtBr) solution (0</ins>.<ins class="diffchange diffchange-inline">5 μg/ml final concentration) was added to </ins>the <ins class="diffchange diffchange-inline">liquid agar, which </ins>was <ins class="diffchange diffchange-inline">then poured </ins>in <ins class="diffchange diffchange-inline">a flat-bed tray with combs. As soon as the agarose solidified, the Running </ins>buffer <ins class="diffchange diffchange-inline">(0</ins>.<ins class="diffchange diffchange-inline">5 x TAE </ins>buffer<ins class="diffchange diffchange-inline">) </ins>was added <ins class="diffchange diffchange-inline">before </ins>the <ins class="diffchange diffchange-inline">DNA in the loading buffer </ins>was <ins class="diffchange diffchange-inline">loaded into </ins>the <ins class="diffchange diffchange-inline">wells and separated electrophoretically</ins>. <ins class="diffchange diffchange-inline">Ethidium bromide intercalates with </ins>the <ins class="diffchange diffchange-inline">DNA’s GC ntss resulting in DNA</ins>-<ins class="diffchange diffchange-inline">EtBr-complex that emits visible light</ins>. <ins class="diffchange diffchange-inline">Therefore, </ins>the <ins class="diffchange diffchange-inline">DNA fragments could be detected on </ins>a <ins class="diffchange diffchange-inline">UV-light tray </ins>at <ins class="diffchange diffchange-inline">265 nm</ins>.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><br></div></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: #ffa; color:black; font-size: smaller;"><div>===<del class="diffchange diffchange-inline">Sequencing</del>===</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">===Colony PCR===</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">performed by the GATC Biotech company</del></div></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">Colony PCRs were performed using Fermentas PCR Master Mix (2x)</ins>, <ins class="diffchange diffchange-inline">containing ''Taq'' DNA Polymerase</ins>.<ins class="diffchange diffchange-inline"><br/></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">Bacterial colonies were picked form an LB/Agar-plate </ins>and <ins class="diffchange diffchange-inline">either resuspended in PCR reaction mix and subsequently used to inoculate LB</ins>-<ins class="diffchange diffchange-inline">medium </ins>with <ins class="diffchange diffchange-inline">appropriate antibiotic, or resuspended in water</ins>. The <ins class="diffchange diffchange-inline">water </ins>was <ins class="diffchange diffchange-inline">then taken as "template" for PCR </ins>and <ins class="diffchange diffchange-inline">inoculation of Minepreps</ins>.<br<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">The PCR was performed using 20 pmol of forward and reverse primer in a total volume of 20 µL.</ins><br<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">PCR conditions were set to recommendations made by Fermentas:</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">{| border</ins>=<ins class="diffchange diffchange-inline">"1" cellpadding</ins>=<ins class="diffchange diffchange-inline">"3" style</ins>=<ins class="diffchange diffchange-inline">"border:solid 1px #AAAAAA; border-collapse:collapse; background-color:#F9F9F9; empty-cells:show;"</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">!Temp !! 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">|95°||03:00||</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">|style</ins>=<ins class="diffchange diffchange-inline">"border-top:solid 1px #000000;"|94°||style</ins>=<ins class="diffchange diffchange-inline">"border-top:solid 1px #000000;border-right:solid 1px #000000;"|00:30||rowspan</ins>=<ins class="diffchange diffchange-inline">"3"|x 30 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">|45°'*'||style="border-right:solid 1px #000000;"|00:30</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">|style="border-bottom:solid 1px #000000;"|72°||style="border-bottom:solid 1px #000000;border-right:solid 1px #000000;"|01:00 /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">|72°||10:00||</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">|4°||8||</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> </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">'*'with 45°C at a minimum of primer annealing temp</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>===Gel extraction===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Gel extraction===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After gel electrophoresis the digested vector and insert have to be purified from the gel. With the help of a UV lamp, the bands were quickly excised from the gel without exposing the DNA too long to UV light. Afterwards the DNA was purified with the QIAquick Gel extraction kit. Three volumes of buffer QG were added to one volume of gel. The gel fragment was dissolved by incubation for 10 min at 50°C. Afterwards one volume of 100% isopropanol was added. The solution was applied on a QIAquick spin column after this has been placed into a provided 2 ml collection tube. By centrifugation for 1 min at 13.000 rpm the DNA was bound to the column. The flow-through was discarded and the column was placed in the same collection tube. To remove all traces of agarose from the column, 500 µl of wash buffer QC was added followed by centrifugation for 1 min at 13.000 rpm. The flow-through was discarded and the column was washed with 750 µl of buffer PE for 1 min at 13.000 rpm. Afterwards the flow-through was discarded. An additional centrifugation for 1 min at 13.000 rpm helped to remove the residual ethanol. The column was placed into a new 1.5 ml microcentrifuge tube and it was eluted with 30 µl of ddH2O.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After gel electrophoresis the digested vector and insert have to be purified from the gel. With the help of a UV lamp, the bands were quickly excised from the gel without exposing the DNA too long to UV light. Afterwards the DNA was purified with the QIAquick Gel extraction kit. Three volumes of buffer QG were added to one volume of gel. The gel fragment was dissolved by incubation for 10 min at 50°C. Afterwards one volume of 100% isopropanol was added. The solution was applied on a QIAquick spin column after this has been placed into a provided 2 ml collection tube. By centrifugation for 1 min at 13.000 rpm the DNA was bound to the column. The flow-through was discarded and the column was placed in the same collection tube. To remove all traces of agarose from the column, 500 µl of wash buffer QC was added followed by centrifugation for 1 min at 13.000 rpm. The flow-through was discarded and the column was washed with 750 µl of buffer PE for 1 min at 13.000 rpm. Afterwards the flow-through was discarded. An additional centrifugation for 1 min at 13.000 rpm helped to remove the residual ethanol. The column was placed into a new 1.5 ml microcentrifuge tube and it was eluted with 30 µl of ddH2O.</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><del style="color: red; font-weight: bold; text-decoration: none;">===Purification of PCR product===</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;">One volume of buffer PBI was added to one volume of the PCR sample mix. The sample was applied to a QIAquick column which has been placed into a provided 2 ml collection tube. It was centrifuged for 1 min at 13.000 rpm and the flow-through was discarded and the column was placed in the same collection tube. After this 750 µl of buffer PE was added to wash the column. It was centrifuged for 1 min at 13.000 rpm. The flow-through was discarded and the column was placed in the same collection tube. It was centrifuged for 1 min at 13.000 rpm. Afterwards the QIAquick column was placed into a new 1.5 ml microcentrifuge tube and it was eluted with 40 µl ddH2O.</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: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">===Agarose Gel Electrophoresis===</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;">Agarose flat-bed gels in various concentrations (0.6–2% agarose in 0.5 x TAE buffer) and sizes were run to separate DNA fragments in an electrical field (10–20 V/cm) for analytical or preparative use. The desired amount of agarose was boiled in 1 x TAE buffer until it was completely dissolved. After it cooled down to approximately 60°C, ethidium bromide (EtBr) solution (0.5 μg/ml final concentration) was added to the liquid agar, which was then poured in a flat-bed tray with combs. As soon as the agarose solidified, the Running buffer (0.5 x TAE buffer) was added before the DNA in the loading buffer was loaded into the wells and separated electrophoretically. Ethidium bromide intercalates with the DNA’s GC ntss resulting in DNA-EtBr-complex that emits visible light. Therefore, the DNA fragments could be detected on a UV-light tray at 265 nm.</del></div></td><td colspan="2"> </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>===Large scale preparation of plasmid DNA===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Large scale preparation of plasmid DNA===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>150 ml LB-Medium with 150 µl ampicillin was inoculated with 50 µl of bacteria culture which grew overnight on a shaker at 37°C. The plasmid DNA was isolated using QiAprep Spin MAxiprep kit from Qiagen and the protocol was followed. The overnight culture was centrifuged for 20 min at 4000 rpm at 4°C using an SLA 1500 Rotor. Afterwards the LB-medium was discarded and the pellet was homogeneously resuspended in 10 ml of precooled Buffer P1. After having added 10 ml of Buffer P2 the mixture was inverted 4-6 times and incubated for 5 min at RT before adding 10 ml of chilled Buffer P3. Thereafter the lysate was poured into a prepared QIAfilter Maxi Cartridge and incubated at RT for 10 min. During this time a QIAGEN-tip 500 was equilibrated by applying 10 ml of Buffer QBT and allowing the column to empty by gravity flow. The cell lysate was filtered into the QIAGEN-tip. The cleared lysate entered the resin by gravity flow and after washing with 2 x 30 ml Buffer QC the Plasmid DNA was eluted with 15 ml Buffer QF. After this the DNA was precipitated by adding 10.5 ml isopropanol and centrifuged at 4,000 rpm for 45 min at 4°C. The supernatant was discarded and the DNA pellet was washed with 5 ml ethanol (70%) and centrifuged at 4,000 rpm for 15 min. After air-drying the pellet the DNA was redissolved in H2O. <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>150 ml LB-Medium with 150 µl ampicillin was inoculated with 50 µl of bacteria culture which grew overnight on a shaker at 37°C. The plasmid DNA was isolated using QiAprep Spin MAxiprep kit from Qiagen and the protocol was followed. The overnight culture was centrifuged for 20 min at 4000 rpm at 4°C using an SLA 1500 Rotor. Afterwards the LB-medium was discarded and the pellet was homogeneously resuspended in 10 ml of precooled Buffer P1. After having added 10 ml of Buffer P2 the mixture was inverted 4-6 times and incubated for 5 min at RT before adding 10 ml of chilled Buffer P3. Thereafter the lysate was poured into a prepared QIAfilter Maxi Cartridge and incubated at RT for 10 min. During this time a QIAGEN-tip 500 was equilibrated by applying 10 ml of Buffer QBT and allowing the column to empty by gravity flow. The cell lysate was filtered into the QIAGEN-tip. The cleared lysate entered the resin by gravity flow and after washing with 2 x 30 ml Buffer QC the Plasmid DNA was eluted with 15 ml Buffer QF. After this the DNA was precipitated by adding 10.5 ml isopropanol and centrifuged at 4,000 rpm for 45 min at 4°C. The supernatant was discarded and the DNA pellet was washed with 5 ml ethanol (70%) and centrifuged at 4,000 rpm for 15 min. After air-drying the pellet the DNA was redissolved in H2O. <br></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>
<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 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;">===Plasmid-DNA isolation===</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;">5 ml LB-Medium with 5 µl ampicillin was inoculated with single colonies which grew overnight on a shaker at 37°C. The plasmid DNA was isolated using QiAprep Spin Miniprep kit from Qiagen and following the manufacturer’s protocol. 4 ml of each overnight culture was pelleted in 2 ml microcentrifuge tubes during two steps of centrifugation at 13.000 rpm. Subsequently the pellet was resuspended in 250 µl of chilled buffer P1. 250 µl of lysis buffer P2 was added and the solution was mixed thoroughly by inverting the tube 4-6 times. After adding 350 µl of the neutralization Buffer N3 the solution was mixed immediately and thoroughly by inverting the tube 4-6 times. Thereafter the mixture was centrifuged. The supernatants were applied to a QIAprep column which was put in a 2 ml collection tube. It was centrifuged for 1 min at 13.000 rpm and the flow-through was discarded. After adding 500 µl of wash buffer PB, it was centrifuged for 1 min at 13.000 rpm and the flow-through was discarded. Once more, it was washed with 750 µl of wash buffer PE. In an additional centrifugation for 1 min at 13.000 rpm the residual wash buffer was removed. The QIAprep column was placed into a clean 1.5 ml microcentrifuge tube and the plasmid DNA was eluted in 30 µl ddH2O.<br></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;"><br></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;"></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;">===Purification of PCR product===</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;">One volume of buffer PBI was added to one volume of the PCR sample mix. The sample was applied to a QIAquick column which has been placed into a provided 2 ml collection tube. It was centrifuged for 1 min at 13.000 rpm and the flow-through was discarded and the column was placed in the same collection tube. After this 750 µl of buffer PE was added to wash the column. It was centrifuged for 1 min at 13.000 rpm. The flow-through was discarded and the column was placed in the same collection tube. It was centrifuged for 1 min at 13.000 rpm. Afterwards the QIAquick column was placed into a new 1.5 ml microcentrifuge tube and it was eluted with 40 µl ddH2O.</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;">===Sequencing===</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;">performed by the GATC Biotech company</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>==Cell culture==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Cell culture==</div></td></tr>
</table>Kleinsorghttp://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Material_Methods&diff=137705&oldid=prevThomasU: /* Pleat reader measurements */2010-10-24T21:36:25Z<p><span class="autocomment">Pleat reader measurements</span></p>
<table style="background-color: white; color:black;">
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Promega GLOMAX 96 Microplate Luminometer</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Promega GLOMAX 96 Microplate Luminometer</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>=== <del class="diffchange diffchange-inline">Pleat </del>reader measurements ===</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>=== <ins class="diffchange diffchange-inline">Plate </ins>reader measurements ===</div></td></tr>
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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>
</table>ThomasUhttp://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Material_Methods&diff=136244&oldid=prevLorenz at 19:01, 24 October 20102010-10-24T19:01:33Z<p></p>
<table style="background-color: white; color:black;">
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|XmaI||25|| 25%|| 50%|| 0%|| 100%|| x</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|XmaI||25|| 25%|| 50%|| 0%|| 100%|| x</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: #ffa; color:black; font-size: smaller;"><div>|<del class="diffchange diffchange-inline">-</del></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 colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"></center></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>NEBuffers are color-coded (NEBuffer 1-yellow, NEBuffer 2-blue, </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>NEBuffers are color-coded (NEBuffer 1-yellow, NEBuffer 2-blue, </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>NEBuffer 3-red, NEBuffer 4-green) and supplied as 10X</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>NEBuffer 3-red, NEBuffer 4-green) and supplied as 10X</div></td></tr>
</table>Lorenzhttp://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Material_Methods&diff=136035&oldid=prevLeaflocke: /* Enzymes */2010-10-24T18:41:31Z<p><span class="autocomment">Enzymes</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| class="wikitable sortable" border="0" style="text-align: left"</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| class="wikitable sortable" border="0" style="text-align: left"</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-bgcolor=#cccccc</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-bgcolor=#cccccc</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>|+ align="top, left"|'''table 3''': Used <del class="diffchange diffchange-inline"><i></del>NEB restriction enzymes<del class="diffchange diffchange-inline"></i> </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|+ align="top, left"|'''table 3''': Used NEB restriction enzymes</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| Name||Number/ID||NEB buffer1||NEB buffer2||NEB buffer3||NEB buffer4||BSA</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| Name||Number/ID||NEB buffer1||NEB buffer2||NEB buffer3||NEB buffer4||BSA</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>
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<td colspan="2" class="diff-lineno">Line 68:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|BglI||-||50%||75%||100%||50%|| </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|BglI||-||50%||75%||100%||50%|| </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: #ffa; color:black; font-size: smaller;"><div>|BglII||13|| 10%|| 75%|| 100%|| 10%<del class="diffchange diffchange-inline">||</del>|| </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|BglII||13|| 10%|| 75%|| 100%|| 10%||</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: #ffa; color:black; font-size: smaller;"><div>|BsrGI||-|| 25%|| 100%|| 10%|| 100%<del class="diffchange diffchange-inline">||</del>||x</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|BsrGI||-|| 25%|| 100%|| 10%|| 100%||x</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>|DpnI ||6|| 100%|| 100%|| 75%|| 100%|| </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|DpnI ||6|| 100%|| 100%|| 75%|| 100%|| </div></td></tr>
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<td colspan="2" class="diff-lineno">Line 112:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|SfcI ||19|| 75%|| 50%|| 10%|| 100%|| x</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|SfcI ||19|| 75%|| 50%|| 10%|| 100%|| x</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: #ffa; color:black; font-size: smaller;"><div>|SfcI||-|| <del class="diffchange diffchange-inline"> </del>75%|| 50%|| 10%|| 100%|| x</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|SfcI||-||75%|| 50%|| 10%|| 100%|| x</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>|SpeI ||-|| 75%|| 100%|| 25%|| 100%|| x</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|SpeI ||-|| 75%|| 100%|| 25%|| 100%|| x</div></td></tr>
</table>Leaflockehttp://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Material_Methods&diff=135974&oldid=prevLeaflocke: /* Enzymes */2010-10-24T18:35:34Z<p><span class="autocomment">Enzymes</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|BglI||-||50%||75%||100%||50%|| </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|BglI||-||50%||75%||100%||50%|| </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: #ffa; color:black; font-size: smaller;"><div>|BglII||13 10%|| 75%|| 100%|| 10%|||| </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|BglII||13<ins class="diffchange diffchange-inline">|| </ins>10%|| 75%|| 100%|| 10%|||| </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: #ffa; color:black; font-size: smaller;"><div>|BsrGI <del class="diffchange diffchange-inline"> </del>25%|| 100%|| 10%|| 100%||||x</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|BsrGI<ins class="diffchange diffchange-inline">||-|| </ins>25%|| 100%|| 10%|| 100%||||x</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: #ffa; color:black; font-size: smaller;"><div>|DpnI 6 100%|| 100%|| 75%|| 100%|| </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|DpnI <ins class="diffchange diffchange-inline">||</ins>6<ins class="diffchange diffchange-inline">|| </ins>100%|| 100%|| 75%|| 100%|| </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: #ffa; color:black; font-size: smaller;"><div>|DpnI <del class="diffchange diffchange-inline"> </del>100%|| 100%|| 75%|| 100%|| </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|DpnI <ins class="diffchange diffchange-inline">||-|| </ins>100%|| 100%|| 75%|| 100%|| </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: #ffa; color:black; font-size: smaller;"><div>|EcoRI <del class="diffchange diffchange-inline"> </del>100%|| 100%|| 100%|| 100%|| </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|EcoRI <ins class="diffchange diffchange-inline">||-|| </ins>100%|| 100%|| 100%|| 100%|| </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: #ffa; color:black; font-size: smaller;"><div>|EcoRV <del class="diffchange diffchange-inline"> </del>50%|| 75%|| 100%|| 50%|| x</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|EcoRV <ins class="diffchange diffchange-inline">||-|| </ins>50%|| 75%|| 100%|| 50%|| x</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: #ffa; color:black; font-size: smaller;"><div>|FseI <del class="diffchange diffchange-inline"> </del>100%|| 75%|| 0%|| 100%|| </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|FseI <ins class="diffchange diffchange-inline">||-|| </ins>100%|| 75%|| 0%|| 100%|| </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: #ffa; color:black; font-size: smaller;"><div>|HindIII 10 50%|| 100%|| 10%|| 50%|| </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|HindIII<ins class="diffchange diffchange-inline">||-|| </ins>10 50%|| 100%|| 10%|| 50%|| </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: #ffa; color:black; font-size: smaller;"><div>|HindIII <del class="diffchange diffchange-inline"> </del>50%|| 100%|| 10%|| 50%|| </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|HindIII<ins class="diffchange diffchange-inline">||-||</ins>50%|| 100%|| 10%|| 50%|| </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: #ffa; color:black; font-size: smaller;"><div>|KpnI 11 100%|| 75%|| 0%|| 50%|| x</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|KpnI <ins class="diffchange diffchange-inline">||</ins>11<ins class="diffchange diffchange-inline">|| </ins>100%|| 75%|| 0%|| 50%|| x</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: #ffa; color:black; font-size: smaller;"><div>|MfeI-HF™ 75%|| 50%|| 10%|| 100%|| </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|MfeI-HF™<ins class="diffchange diffchange-inline">||-|| </ins>75%|| 50%|| 10%|| 100%|| </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: #ffa; color:black; font-size: smaller;"><div>|NarI 26 100%|| 75%|| 75%|| 100%|| </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|NarI <ins class="diffchange diffchange-inline">||</ins>26<ins class="diffchange diffchange-inline">|| </ins>100%|| 75%|| 75%|| 100%|| </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>|NcoI ||12|| 100%|| 100%|| 100%|| 100%|| </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|NcoI ||12|| 100%|| 100%|| 100%|| 100%|| </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|SacI ||16|| 100%|| 50%|| 10%|| 100%|| </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|SacI ||16|| 100%|| 50%|| 10%|| 100%|| </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: #ffa; color:black; font-size: smaller;"><div>|ScaI ||18|| not not 100%|| not </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|ScaI ||18|| not<ins class="diffchange diffchange-inline">||</ins>not<ins class="diffchange diffchange-inline">||</ins>100%||not<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>|-</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>|SfcI ||19|| 75%|| 50%|| 10%|| 100%|| x</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|SfcI ||19|| 75%|| 50%|| 10%|| 100%|| x</div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 127:</td>
<td colspan="2" class="diff-lineno">Line 127:</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>|XmaI||25|| 25%|| 50%|| 0%|| 100%|| x</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|XmaI||25|| 25%|| 50%|| 0%|| 100%|| x</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 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 class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>NEBuffers are color-coded (NEBuffer 1-yellow, NEBuffer 2-blue, </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>NEBuffers are color-coded (NEBuffer 1-yellow, NEBuffer 2-blue, </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>NEBuffer 3-red, NEBuffer 4-green) and supplied as 10X</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>NEBuffer 3-red, NEBuffer 4-green) and supplied as 10X</div></td></tr>
</table>Leaflockehttp://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Material_Methods&diff=135889&oldid=prevLeaflocke: /* Enzymes */2010-10-24T18:28:55Z<p><span class="autocomment">Enzymes</span></p>
<table style="background-color: white; color:black;">
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 18:28, 24 October 2010</td>
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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>== Enzymes ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Enzymes ==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">... still about to come.</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><center></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">{| class="wikitable sortable" border="0" style="text-align: left"</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">|-bgcolor=#cccccc</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">|+ align="top, left"|'''table 3''': Used <i>NEB restriction enzymes</i> </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">| Name||Number/ID||NEB buffer1||NEB buffer2||NEB buffer3||NEB buffer4||BSA</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">|AflII||27||50%||100%||25%||100%||x</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">|AgeI||1||100%||50%||10%||75% </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">|ApaI||-||25%||50%||0%||100%||x</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">|AseI||not||75%||100%||not|| </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">|AvrII||100%||100%||50%||100%||</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">|BamHI||2||75%||100%||100%||100%||x</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">|BamHI||-||75%||100%||100%||100%||x</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">|BclI||-||50%||100%||100%||75%|| </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">|BglI||-||50%||75%||100%||50%|| </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">|BglII||13 10%|| 75%|| 100%|| 10%|||| </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">|BsrGI 25%|| 100%|| 10%|| 100%||||x</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">|DpnI 6 100%|| 100%|| 75%|| 100%|| </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">|DpnI 100%|| 100%|| 75%|| 100%|| </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">|EcoRI 100%|| 100%|| 100%|| 100%|| </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">|EcoRV 50%|| 75%|| 100%|| 50%|| x</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">|FseI 100%|| 75%|| 0%|| 100%|| </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">|HindIII 10 50%|| 100%|| 10%|| 50%|| </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">|HindIII 50%|| 100%|| 10%|| 50%|| </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">|KpnI 11 100%|| 75%|| 0%|| 50%|| x</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">|MfeI-HF™ 75%|| 50%|| 10%|| 100%|| </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">|NarI 26 100%|| 75%|| 75%|| 100%|| </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">|NcoI ||12|| 100%|| 100%|| 100%|| 100%|| </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">|NdeI ||-|| 75%|| 100%|| 75%|| 100%|| </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">|NheI ||-|| 100%|| 100%|| 10%|| 100%|| x</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">|NotI ||14|| 0%|| 50%|| 100%|| 25%|| </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">|NsiI ||-|| 10%|| 75%|| 100%|| 25%|| </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">|PciI ||-|| 50%|| 75%|| 100%|| 50%|| x</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">|PstI ||15|| 75%|| 75%|| 100%|| 50%|| </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">|SacI ||16|| 100%|| 50%|| 10%|| 100%|| </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">|ScaI ||18|| not not 100%|| not </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">|SfcI ||19|| 75%|| 50%|| 10%|| 100%|| x</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">|SfcI||-|| 75%|| 50%|| 10%|| 100%|| x</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">|SpeI ||-|| 75%|| 100%|| 25%|| 100%|| x</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">|SphI ||-|| 100%|| 100%|| 50%|| 100%|| </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">|SspI ||-|| 50%|| 100%|| 50%|| 50%|| </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">|XbaI ||23 ||0%|| 100%|| 75%|| 100%|| x</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">|XbaI ||56|| 0%|| 100%|| 75%|| 100%|| x</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">|XhoI||24|| 75%|| 100%|| 100%|| 100%|| x</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">|XmaI||25|| 25%|| 50%|| 0%|| 100%|| x</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">NEBuffers are color-coded (NEBuffer 1-yellow, NEBuffer 2-blue, </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">NEBuffer 3-red, NEBuffer 4-green) and supplied as 10X</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 class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Bacteria ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Bacteria ==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center></div></td></tr>
</table>Leaflockehttp://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Material_Methods&diff=135147&oldid=prevLaura Nadine: /* Buffers */2010-10-24T17:34:40Z<p><span class="autocomment">Buffers</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== Buffers ====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== Buffers ====</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>*'''Coating Buffer''' (0.1M NaHCO<sub>3<, 0.1M Na<sub>2</sub>CO<sub>3</sub>, pH 9.5) </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>*'''Coating Buffer''' (0.1M NaHCO<sub>3<<ins class="diffchange diffchange-inline">/sub></ins>, 0.1M Na<sub>2</sub>CO<sub>3</sub>, pH 9.5) </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>*'''Diluting/Blocking Buffer (db)''' (0.25 ml Tween 20, 30 g BSA, add 1x PBS to 500 ml)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*'''Diluting/Blocking Buffer (db)''' (0.25 ml Tween 20, 30 g BSA, add 1x PBS to 500 ml)</div></td></tr>
</table>Laura Nadinehttp://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Material_Methods&diff=135140&oldid=prevLaura Nadine: /* Buffers */2010-10-24T17:34:11Z<p><span class="autocomment">Buffers</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== Buffers ====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== Buffers ====</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>*'''Coating Buffer''' (0.<del class="diffchange diffchange-inline">68 g sodium carbonate, </del>3<del class="diffchange diffchange-inline">.675 g sodium bicarbonate</del>, <del class="diffchange diffchange-inline">pH 9</del>.<del class="diffchange diffchange-inline">2 with 5N HCl, ad 500 ml ddH</del><sub>2</sub><del class="diffchange diffchange-inline">O</del>)</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>*'''Coating Buffer''' (0.<ins class="diffchange diffchange-inline">1M NaHCO<sub></ins>3<ins class="diffchange diffchange-inline"><</ins>, <ins class="diffchange diffchange-inline">0</ins>.<ins class="diffchange diffchange-inline">1M Na</ins><sub>2</sub><ins class="diffchange diffchange-inline">CO<sub>3</sub>, pH 9.5</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>*'''Diluting/Blocking Buffer (db)''' (0.25 ml Tween 20, 30 g BSA, add 1x PBS to 500 ml)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*'''Diluting/Blocking Buffer (db)''' (0.25 ml Tween 20, 30 g BSA, add 1x PBS to 500 ml)</div></td></tr>
</table>Laura Nadinehttp://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Material_Methods&diff=135058&oldid=prevLaura Nadine: /* Procedure */2010-10-24T17:28:40Z<p><span class="autocomment">Procedure</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* incubate 1 hour at 37°C</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* incubate 1 hour at 37°C</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* wash five times</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* wash five times</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">#* make substrate: per plate 2 OPD tablets into 15 ml Sodium Citrate</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#* add 100 µl substrate per well</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#* add <del class="diffchange diffchange-inline">3.2 µl of H<sub>2</sub>O<sub>2</sub></del></div></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: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">#* </del>100 µl substrate per well</div></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;"><div>#* incubate at room temperature for 5 - 10 min</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#* incubate at room temperature for 5 - 10 min</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#* stop with <del class="diffchange diffchange-inline">50 µl 2.5 M </del>H<sub>2</sub>SO<sub>4</sub></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#* stop with <ins class="diffchange diffchange-inline">100µl 2M </ins>H<sub>2</sub>SO<sub>4</sub> <ins class="diffchange diffchange-inline">per well</ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#* read at <del class="diffchange diffchange-inline">490 </del>nm wavelength</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#* read <ins class="diffchange diffchange-inline">absorbance </ins>at <ins class="diffchange diffchange-inline">450 </ins>nm wavelength</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">==== Buffers ====</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">*'''Coating Buffer''' (0.68 g sodium carbonate, 3.675 g sodium bicarbonate, pH 9.2 with 5N HCl, ad 500 ml ddH<sub>2</sub>O)</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">*'''Diluting/Blocking Buffer (db)''' (0.25 ml Tween 20, 30 g BSA, add 1x PBS to 500 ml)</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">*'''Wash Buffer''' (0.5 ml Tween 20, 1L PBS 1x)</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> </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">:'''Calibration'''</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">* 960 µl db + 40 µl pooled plasma = 200 ng/ml</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 500 µl of above into 500 µl of db = 100 ng/ml</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">* serial dilutions continued down to 1.5625 ng/ml</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">:'''Detection reagents'''</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">* TMB Substrate Kit (Pierce, Thermo Scientific)</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">* 2M H<sub>2</sub>SO<sub>4</sub></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>=== Microscopy ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== Microscopy ===</div></td></tr>
</table>Laura Nadine