User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/14: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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| colspan="2"| | | colspan="2"| | ||
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| bgcolor=#cfcfcf | (x9) | | bgcolor=#cfcfcf | (x9) | ||
| rowspan="7" | Expected:<br>1-8. MV10 = 5191 | | rowspan="7" | Expected:<br>1-8. MV10 = 5191 | ||
| rowspan="7" | [[Image: | | rowspan="7" | [[Image:KAH041415_gel1.jpg|250px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
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| Template || 0.2 || 1.8 | | Template || 0.2 || 1.8 | ||
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| bgcolor=#cfcfcf | Rxn3,4 | | bgcolor=#cfcfcf | Rxn3,4 | ||
| rowspan="7" | Expected:<br>1,2. Gal4DB-mCh = 1170<br>3,4. ATF2 = 906<br> | | rowspan="7" | Expected:<br>1,2. Gal4DB-mCh = 1170<br>3,4. ATF2 = 906<br> | ||
| rowspan="7" | [[Image: | | rowspan="7" | [[Image:KAH041415_gel2.jpg|200px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
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| Template || 0.2 || 3.0 | | Template || 0.2 || 3.0 | ||
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* Gal4DB-mCh - success! | * Gal4DB-mCh - success! | ||
** purify with Sigma PCR purification kit | ** purify with Sigma PCR purification kit | ||
** Elute | ** Elute & back-elute w/ 25 μL elution sln. | ||
* ATF2 - failed | * ATF2 - failed | ||
** Try again with 1:10 and undiluted template | ** Try again with 1:10 and undiluted template | ||
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| Sample || OD260 || 260/280 || ng/μL | | Sample || OD260 || 260/280 || ng/μL | ||
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| 1. 5'p-Gal4DB-mCh || | | 1. 5'p-Gal4DB-mCh || 0.01 || 1.23 || 9.68 | ||
|} | |} | ||
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| bgcolor=#cfcfcf | (x9) | | bgcolor=#cfcfcf | (x9) | ||
| rowspan="7" | Expected:<br>1-8. MV10 = 5191 | | rowspan="7" | Expected:<br>1-8. MV10 = 5191 | ||
| rowspan="7" | [[Image: | | rowspan="7" | [[Image:KAH041415_gel3.jpg|250px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
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| Template || 0.2 || 1.8 | | Template || 0.2 || 1.8 | ||
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| Phusion pol. || 0.5 || 4.5 | | Phusion pol. || 0.5 || 4.5 | ||
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| 5x GC buffer || 5.0 || 45.0 | | 5x '''GC buffer''' || 5.0 || 45.0 | ||
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| DMSO || 1.5 || 7.5 | | '''DMSO''' || 1.5 || 7.5 | ||
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| dH<sub>2</sub>O || 39.8 || 358.2 | | dH<sub>2</sub>O || 39.8 || 358.2 | ||
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Conclusion | Conclusion | ||
* | * Failed - try using XbaI-linearized vector as template | ||
** XbaI cuts in between the two primers, which face outward from the XbaI site | |||
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| bgcolor=#cfcfcf | Rxn3,4 | | bgcolor=#cfcfcf | Rxn3,4 | ||
| rowspan="7" | Expected:<br>1,2. Gal4DB-mCh = 1170<br>3,4. ATF2 = 906<br> | | rowspan="7" | Expected:<br>1,2. Gal4DB-mCh = 1170<br>3,4. ATF2 = 906<br> | ||
| rowspan="7" | | | rowspan="7" | No visible product in any lanes | ||
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| Template || 0.5 || 2.0 | | Template || 0.5 || 2.0 | ||
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Conclusion | Conclusion | ||
* | * Failed - order ATF2 cDNA, use as new template for PCR | ||
Latest revision as of 00:54, 27 September 2017
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04/14/15
LCR Protocol Development
Program: Phusion (block A) - edited to include 65°C - 55°C annealing gradient
Conclusion
Gal4DB-mCh - plasmid
Program: Phusion (block B)
Conclusion
Program: Phusion (block A) - edited to include 65°C - 55°C annealing gradient
Conclusion
Program: Phusion (block B)
Conclusion
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