User:Karmella Haynes/Notebook/BioBrick cloning/2013/04/12: 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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== | ==04/12/13== | ||
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* | * Chem. Competent cell prep: CaCl wash; 15% glycerol/CaCl resuspension (used 30 mL instead of 20 mL); incubate in cold room on ice o/n | ||
* | * MV9 building: colony PCR | ||
---- | ---- | ||
'''MV9 building: colony PCR''' | |||
* Pick 8 colonies from plates 1, 2, 3, and 4; make a mini-streak library, then wash pipette tip in PCR master mix | |||
* Pick 4 colonies from plate 5 (neg. ctrl); do not add to streak library; include in PCR as neg ctrl.s | |||
* Primers for MV2 vector: DD122B (fwd), DD123B (rev); see [http://partsregistry.org/Part:BBa_J176122 BBa_J176122] | |||
{| {{table}} cellspacing="3" <!-- | {| {{table}} cellspacing="3" <!-- PCR rxn. table --> | ||
|- valign="top" | |- valign="top" | ||
| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| rowspan="7" | < | | bgcolor=#cfcfcf | x38 | ||
| rowspan="7" | Expected:<br>MV2 (empty) = 177<br>MV9 (single insert) = 253<br>extra inserts = +76 | |||
| rowspan="7" | [[Image:KAH041513_gel1.jpg|270px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|- | |- | ||
| | | DNA (colony) || --- || --- | ||
|- | |- | ||
| | | 10 μM primer 1 || 1.0 || 38.0 | ||
|- | |- | ||
| | | 10 μM primer 2 || 1.0 || 38.0 | ||
|- | |- | ||
| | | 2x GoTaq green || 10.0 || 380.0 | ||
| | |||
|- | |- | ||
| | | dH<sub>2</sub>O || 8.0 || 304.0 | ||
|- | |- | ||
| | | || 20 μL || | ||
|} | |} | ||
Thermal cycling | |||
* 95°C, 3 min. | |||
* [95°C, 15 sec; 57°C, 15 sec; 72°C, 15 sec] x30 | |||
* 72°C, 3 min. | |||
* 4°C, ∞ | |||
Latest revision as of 22:37, 26 September 2017
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04/12/13
MV9 building: colony PCR
Thermal cycling
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