Haynes:TypeIIS Assembly - Revision history

http://openwetware.org/index.php?title=Haynes:TypeIIS_Assembly&feed=atom&action=history Haynes:TypeIIS Assembly - Revision history 2013-05-19T14:46:36Z Revision history for this page on the wiki MediaWiki 1.13.2 http://openwetware.org/index.php?title=Haynes:TypeIIS_Assembly&diff=679110&oldid=prev Karmella Haynes at 18:20, 25 February 2013 2013-02-25T18:20:19Z

<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 18:20, 25 February 2013</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 55:</td> <td colspan="2" class="diff-lineno">Line 55:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| <br>'''Dilute the purified PCR product to 20 fmol/μL'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| <br>'''Dilute the purified PCR product to 20 fmol/μL'''</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Measure ng/μL of the purified sample.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Measure ng/μL of the purified sample.</div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = '''length in bp''' ÷ '''measured ng/μL''' * <del class="diffchange diffchange-inline">(</del>20 fmols/μL * 650 fg/fmol ÷ 1,000,000 fg/ng <del class="diffchange diffchange-inline">) </del>* 20 μL final volume  </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = '''length in bp''' ÷ '''measured ng/μL''' * <ins class="diffchange diffchange-inline">''</ins>20 fmols/μL * 650 fg/fmol ÷ 1,000,000 fg/ng<ins class="diffchange diffchange-inline">'' </ins>* 20 μL final volume  </div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>** Formula: x = '''length in bp''' ÷ '''measured ng/μL''' * 0.013 <del class="diffchange diffchange-inline">* '</del>''20<del class="diffchange diffchange-inline">'''</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>** Formula: x = '''length in bp''' ÷ '''measured ng/μL''' * <ins class="diffchange diffchange-inline">''</ins>0.013'' <ins class="diffchange diffchange-inline">* </ins>20</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** Note: final volume can be changed by changing the last number from 20 to something else.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** Note: final volume can be changed by changing the last number from 20 to something else.</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>

Karmella Haynes http://openwetware.org/index.php?title=Haynes:TypeIIS_Assembly&diff=679109&oldid=prev Karmella Haynes at 18:19, 25 February 2013 2013-02-25T18:19:29Z

<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 18:19, 25 February 2013</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 55:</td> <td colspan="2" class="diff-lineno">Line 55:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| <br>'''Dilute the purified PCR product to 20 fmol/μL'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| <br>'''Dilute the purified PCR product to 20 fmol/μL'''</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Measure ng/μL of the purified sample.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Measure ng/μL of the purified sample.</div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = <del class="diffchange diffchange-inline">20 μL final volume * 20 fmols/μL * </del>'''length in bp''' * 650 fg/fmol ÷ 1,000,000 fg/ng <del class="diffchange diffchange-inline"> ÷ '''measured ng/</del>μL<del class="diffchange diffchange-inline">'''</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = '''length in bp''' <ins class="diffchange diffchange-inline">÷ '''measured ng/μL''' * (20 fmols/μL </ins>* 650 fg/fmol ÷ 1,000,000 fg/ng <ins class="diffchange diffchange-inline">) * 20 </ins>μL <ins class="diffchange diffchange-inline">final volume </ins></div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>** Formula: x = '''length in bp''' ÷ '''measured ng/μL''' * 0.<del class="diffchange diffchange-inline">26</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>** Formula: x = '''length in bp''' ÷ '''measured ng/μL''' * 0.<ins class="diffchange diffchange-inline">013 * '''20'''</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">** Note: final volume can be changed by changing the last number from 20 to something else.</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>| [[Image:Haynes_TIIS_fig7.png|250px|Figure 7]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| [[Image:Haynes_TIIS_fig7.png|250px|Figure 7]]</div></td></tr>  </table>

Karmella Haynes http://openwetware.org/index.php?title=Haynes:TypeIIS_Assembly&diff=671372&oldid=prev Karmella Haynes at 15:08, 29 January 2013 2013-01-29T15:08:55Z

<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 15:08, 29 January 2013</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 91:</td> <td colspan="2" class="diff-lineno">Line 91:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><font size=3>'''Bacterial transformation'''</font></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><font size=3>'''Bacterial transformation'''</font></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Add total volume (10.0 μL) to 50 μL chemically competent cells  (e.g., BL21) in a 2.0 mL tube.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Add total volume (10.0 μL) to 50 μL chemically competent cells  (e.g., BL21) in a 2.0 mL tube.</div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* Incubate on ice for 2 min., heat shock at 42°C for exactly <del class="diffchange diffchange-inline">90 </del>sec., immediately place on ice.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* Incubate on ice for 2 min., heat shock at 42°C for exactly <ins class="diffchange diffchange-inline">45 </ins>sec., immediately place on ice.</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Add 800 μL sterile SOC medium.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Add 800 μL sterile SOC medium.</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Grow with shaking at 37°C for 30 min.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Grow with shaking at 37°C for 30 min.</div></td></tr>  </table>

Karmella Haynes http://openwetware.org/index.php?title=Haynes:TypeIIS_Assembly&diff=668957&oldid=prev Karmella Haynes at 01:32, 20 January 2013 2013-01-20T01:32:03Z

<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 01:32, 20 January 2013</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 58:</td> <td colspan="2" class="diff-lineno">Line 58:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** Formula: x = '''length in bp''' ÷ '''measured ng/μL''' * 0.26</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** Formula: x = '''length in bp''' ÷ '''measured ng/μL''' * 0.26</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">IMage</del>:Haynes_TIIS_fig7.png|250px|Figure 7]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>| [[<ins class="diffchange diffchange-inline">Image</ins>:Haynes_TIIS_fig7.png|250px|Figure 7]]</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| '''Perform BsmBI/ T4 ligase mediated assembly'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| '''Perform BsmBI/ T4 ligase mediated assembly'''</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;">* BsmBI cuts the DNA fragments and creates complementary overhangs.</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;">* Complementary sticky ends anneal via base pairing.</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;">* T4 ligase seals gaps in the phosphodiester DNA backbone.</ins></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| {{table}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| {{table}}</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>

Karmella Haynes http://openwetware.org/index.php?title=Haynes:TypeIIS_Assembly&diff=668956&oldid=prev Karmella Haynes at 01:24, 20 January 2013 2013-01-20T01:24:20Z

<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 01:24, 20 January 2013</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 49:</td> <td colspan="2" class="diff-lineno">Line 49:</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>| [[Image:Haynes_TIIS_fig6.png|250px|Figure 6]] || Run separate 50 μL PCR reactions for each part. If you are using plasmid DNA as a template, use no more than 10 ng in order to minimize carry-over into the final bacterial transformation step. Check 10 μL of the reaction on and agarose gel. Purify the remaining 40 μL of PCR products using a Zymo clean and Concentrator kit (or similar PCR clean up kit).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| [[Image:Haynes_TIIS_fig6.png|250px|Figure 6]] || Run separate 50 μL PCR reactions for each part. If you are using plasmid DNA as a template, use no more than 10 ng in order to minimize carry-over into the final bacterial transformation step. Check 10 μL of the reaction on and agarose gel. Purify the remaining 40 μL of PCR products using a Zymo clean and Concentrator kit (or similar PCR clean up kit).</div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"><br><br></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: #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>|- 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>| <font size=3>'''Digestion/ ligation reaction'''</font></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>| <ins class="diffchange diffchange-inline"><br></ins><font size=3>'''Digestion/ ligation reaction'''</font></div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>| '''Dilute the purified PCR product to 20 fmol/μL'''</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>| <ins class="diffchange diffchange-inline"><br></ins>'''Dilute the purified PCR product to 20 fmol/μL'''</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Measure ng/μL of the purified sample.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Measure ng/μL of the purified sample.</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = 20 μL final volume * 20 fmols/μL * '''length in bp''' * 650 fg/fmol ÷ 1,000,000 fg/ng  ÷ '''measured ng/μL'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = 20 μL final volume * 20 fmols/μL * '''length in bp''' * 650 fg/fmol ÷ 1,000,000 fg/ng  ÷ '''measured ng/μL'''</div></td></tr> <tr><td colspan="2" class="diff-lineno">Line 86:</td> <td colspan="2" class="diff-lineno">Line 86:</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>'''Bacterial transformation'''</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><font size=3></ins>'''Bacterial transformation'''<ins class="diffchange diffchange-inline"></font></ins></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Add total volume (10.0 μL) to 50 μL chemically competent cells  (e.g., BL21) in a 2.0 mL tube.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Add total volume (10.0 μL) to 50 μL chemically competent cells  (e.g., BL21) in a 2.0 mL tube.</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Incubate on ice for 2 min., heat shock at 42°C for exactly 90 sec., immediately place on ice.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Incubate on ice for 2 min., heat shock at 42°C for exactly 90 sec., immediately place on ice.</div></td></tr>  </table>

Karmella Haynes http://openwetware.org/index.php?title=Haynes:TypeIIS_Assembly&diff=668955&oldid=prev Karmella Haynes at 01:18, 20 January 2013 2013-01-20T01:18:00Z

<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 01:18, 20 January 2013</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 58:</td> <td colspan="2" class="diff-lineno">Line 58:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** Formula: x = '''length in bp''' ÷ '''measured ng/μL''' * 0.26</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** Formula: x = '''length in bp''' ÷ '''measured ng/μL''' * 0.26</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">image</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>| <ins class="diffchange diffchange-inline">[[IMage:Haynes_TIIS_fig7.png|250px|Figure 7]]</ins></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| '''Perform BsmBI/ T4 ligase mediated assembly'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| '''Perform BsmBI/ T4 ligase mediated assembly'''</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| {{table}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| {{table}}</div></td></tr>  </table>

Karmella Haynes http://openwetware.org/index.php?title=Haynes:TypeIIS_Assembly&diff=668953&oldid=prev Karmella Haynes at 00:41, 20 January 2013 2013-01-20T00:41:27Z

<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 00:41, 20 January 2013</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 49:</td> <td colspan="2" class="diff-lineno">Line 49:</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>| [[Image:Haynes_TIIS_fig6.png|250px|Figure 6]] || Run separate 50 μL PCR reactions for each part. If you are using plasmid DNA as a template, use no more than 10 ng in order to minimize carry-over into the final bacterial transformation step. Check 10 μL of the reaction on and agarose gel. Purify the remaining 40 μL of PCR products using a Zymo clean and Concentrator kit (or similar PCR clean up kit).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| [[Image:Haynes_TIIS_fig6.png|250px|Figure 6]] || Run separate 50 μL PCR reactions for each part. If you are using plasmid DNA as a template, use no more than 10 ng in order to minimize carry-over into the final bacterial transformation step. Check 10 μL of the reaction on and agarose gel. Purify the remaining 40 μL of PCR products using a Zymo clean and Concentrator kit (or similar PCR clean up kit).</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><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 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>| <font size=3>'''Digestion/ ligation reaction'''</font></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| <font size=3>'''Digestion/ ligation reaction'''</font></div></td></tr>  </table>

Karmella Haynes http://openwetware.org/index.php?title=Haynes:TypeIIS_Assembly&diff=668952&oldid=prev Karmella Haynes at 00:40, 20 January 2013 2013-01-20T00:40:50Z

<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 00:40, 20 January 2013</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 14:</td> <td colspan="2" class="diff-lineno">Line 14:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| width=800px cellpadding=5</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| width=800px cellpadding=5</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>| <font size=<del class="diffchange diffchange-inline">4</del>>'''Use PCR to prepare the parts'''</font></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>| <font size=<ins class="diffchange diffchange-inline">3</ins>>'''Use PCR to prepare the parts'''</font></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>* Multiple parts can be assembled in one step.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Multiple parts can be assembled in one step.</div></td></tr> <tr><td colspan="2" class="diff-lineno">Line 50:</td> <td colspan="2" class="diff-lineno">Line 50:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| [[Image:Haynes_TIIS_fig6.png|250px|Figure 6]] || Run separate 50 μL PCR reactions for each part. If you are using plasmid DNA as a template, use no more than 10 ng in order to minimize carry-over into the final bacterial transformation step. Check 10 μL of the reaction on and agarose gel. Purify the remaining 40 μL of PCR products using a Zymo clean and Concentrator kit (or similar PCR clean up kit).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| [[Image:Haynes_TIIS_fig6.png|250px|Figure 6]] || Run separate 50 μL PCR reactions for each part. If you are using plasmid DNA as a template, use no more than 10 ng in order to minimize carry-over into the final bacterial transformation step. Check 10 μL of the reaction on and agarose gel. Purify the remaining 40 μL of PCR products using a Zymo clean and Concentrator kit (or similar PCR clean up kit).</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>| <font size=<del class="diffchange diffchange-inline">4</del>>'''Digestion/ ligation reaction'''</font></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>| <font size=<ins class="diffchange diffchange-inline">3</ins>>'''Digestion/ ligation reaction'''</font></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| '''Dilute the purified PCR product to 20 fmol/μL'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| '''Dilute the purified PCR product to 20 fmol/μL'''</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Measure ng/μL of the purified sample.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Measure ng/μL of the purified sample.</div></td></tr>  </table>

Karmella Haynes http://openwetware.org/index.php?title=Haynes:TypeIIS_Assembly&diff=668951&oldid=prev Karmella Haynes at 00:40, 20 January 2013 2013-01-20T00:40:27Z

<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 00:40, 20 January 2013</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 14:</td> <td colspan="2" class="diff-lineno">Line 14:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| width=800px cellpadding=5</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| width=800px cellpadding=5</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>| '''Use PCR to prepare the parts'''</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>| <ins class="diffchange diffchange-inline"><font size=4></ins>'''Use PCR to prepare the parts'''<ins class="diffchange diffchange-inline"></font></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>* Multiple parts can be assembled in one step.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Multiple parts can be assembled in one step.</div></td></tr> <tr><td colspan="2" class="diff-lineno">Line 50:</td> <td colspan="2" class="diff-lineno">Line 50:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| [[Image:Haynes_TIIS_fig6.png|250px|Figure 6]] || Run separate 50 μL PCR reactions for each part. If you are using plasmid DNA as a template, use no more than 10 ng in order to minimize carry-over into the final bacterial transformation step. Check 10 μL of the reaction on and agarose gel. Purify the remaining 40 μL of PCR products using a Zymo clean and Concentrator kit (or similar PCR clean up kit).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| [[Image:Haynes_TIIS_fig6.png|250px|Figure 6]] || Run separate 50 μL PCR reactions for each part. If you are using plasmid DNA as a template, use no more than 10 ng in order to minimize carry-over into the final bacterial transformation step. Check 10 μL of the reaction on and agarose gel. Purify the remaining 40 μL of PCR products using a Zymo clean and Concentrator kit (or similar PCR clean up kit).</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>| '''Digestion/ ligation reaction'''</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>| <ins class="diffchange diffchange-inline"><font size=4></ins>'''Digestion/ ligation reaction'''<ins class="diffchange diffchange-inline"></font></ins></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| '''Dilute the purified PCR product to 20 fmol/μL'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| '''Dilute the purified PCR product to 20 fmol/μL'''</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Measure ng/μL of the purified sample.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Measure ng/μL of the purified sample.</div></td></tr>  </table>

Karmella Haynes http://openwetware.org/index.php?title=Haynes:TypeIIS_Assembly&diff=668950&oldid=prev Karmella Haynes at 00:39, 20 January 2013 2013-01-20T00:39:29Z

<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 00:39, 20 January 2013</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 50:</td> <td colspan="2" class="diff-lineno">Line 50:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| [[Image:Haynes_TIIS_fig6.png|250px|Figure 6]] || Run separate 50 μL PCR reactions for each part. If you are using plasmid DNA as a template, use no more than 10 ng in order to minimize carry-over into the final bacterial transformation step. Check 10 μL of the reaction on and agarose gel. Purify the remaining 40 μL of PCR products using a Zymo clean and Concentrator kit (or similar PCR clean up kit).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>| [[Image:Haynes_TIIS_fig6.png|250px|Figure 6]] || Run separate 50 μL PCR reactions for each part. If you are using plasmid DNA as a template, use no more than 10 ng in order to minimize carry-over into the final bacterial transformation step. Check 10 μL of the reaction on and agarose gel. Purify the remaining 40 μL of PCR products using a Zymo clean and Concentrator kit (or similar PCR clean up kit).</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>| '''Digestion/ ligation reaction''' <del class="diffchange diffchange-inline">|| &nbsp;</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>| '''Digestion/ ligation reaction'''</div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>|<del class="diffchange diffchange-inline">- valign="top"</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>| <ins class="diffchange diffchange-inline">'''</ins>Dilute the purified PCR product to 20 fmol/μL<ins class="diffchange diffchange-inline">'''</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">| &nbsp; || 1. </del>Dilute the purified PCR product to 20 fmol/μL</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>* Measure ng/μL of the purified sample.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Measure ng/μL of the purified sample.</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = 20 μL final volume * 20 fmols/μL * '''length in bp''' * 650 fg/fmol ÷ 1,000,000 fg/ng  ÷ '''measured ng/μL'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = 20 μL final volume * 20 fmols/μL * '''length in bp''' * 650 fg/fmol ÷ 1,000,000 fg/ng  ÷ '''measured ng/μL'''</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** Formula: x = '''length in bp''' ÷ '''measured ng/μL''' * 0.26</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** Formula: x = '''length in bp''' ÷ '''measured ng/μL''' * 0.26</div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"><br></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 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">| image</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">2. </del>Perform BsmBI/ T4 ligase mediated assembly</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">| '''</ins>Perform BsmBI/ T4 ligase mediated assembly<ins class="diffchange diffchange-inline">'''</ins></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></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| {{table}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| {{table}}</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>

Karmella Haynes