User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/17: 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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==04/ | ==04/17/15== | ||
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* Rene - gRNA plasmids, 5 mL cultures (11 total) | * Rene - gRNA plasmids, 5 mL cultures (11 total) | ||
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* Stage 1 - insert Regulator ORFS | * Stage 1 - insert Regulator ORFS | ||
** Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites) | ** Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites) | ||
** Insert ORF(E/X) into Vector ( | ** Insert ORF(E/X) into Vector (E/X). see [https://benchling.com/s/VFNzuauP/edit Vector plasmid map] | ||
* Stage 2 - insert Promoters | * Stage 2 - insert Promoters | ||
** Cut & dephos promoters with E/X | ** Cut & dephos promoters with E/X | ||
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| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| bgcolor=#cfcfcf | Mix (x5) | | bgcolor=#cfcfcf | Mix (x5) | ||
| rowspan="7" | <u>Expected:</u><br>1. | | rowspan="7" | <u>Expected:</u><br>1. AubR = 851<br>2. BjaR = 776<br>3. BraR = 776<br>4. RpaR = 779<br> | ||
| rowspan="7" | | | rowspan="7" | [[Image:KAH041715_gel1.jpg|200px|Hover name]]<br>5 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
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| DNA(plasmid) || 0.5 μL || --- | | DNA(plasmid) || 0.5 μL || --- | ||
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Program: Phusion (block B) | Program: Phusion (block B) | ||
* 98°C, 3 min | * 98°C, 3 min | ||
* 35x[98°C, 10 sec; 66°C, 30 sec; 72°C, | * 35x[98°C, 10 sec; 66°C, 30 sec; 72°C, 60 sec] | ||
* 72°C, 10 min | * 72°C, 10 min | ||
* 4°C ∞ | * 4°C ∞ | ||
Conclusion | Conclusion | ||
* | * All four worked. Proceed to next step | ||
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| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Rxn1,2 | | bgcolor=#cfcfcf | Rxn1,2 | ||
| rowspan="7" | <u>Expected:</u><br>1 | | rowspan="7" | <u>Expected:</u><br>1,2. MV10 = 5191 | ||
| rowspan="7" | | | rowspan="7" | [[Image:KAH041715_gel2.jpg|150px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
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| DNA(clean linear) || 1.0 μL | | DNA(clean linear) || 1.0 μL | ||
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Program: Phusion (block | Program: Phusion (block A) | ||
* 98°C, 3 min | * 98°C, 3 min | ||
* 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 2.5 min] | * 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 2.5 min] | ||
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Conclusion | Conclusion | ||
* | * Big improvement. Keep this for potential cloning | ||
* Also try again with higher annealing temp | |||
Latest revision as of 00:55, 27 September 2017
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04/17/15
Ryan - Receiver plasmids, assembly
Conclusion
LCR Development
Conclusion
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