User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/11: Difference between revisions
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* Cancer PcTF - cultures for KAH87_MV10 clone #1 from streak library (clone #3 is wrong) | * Cancer PcTF - cultures for KAH87_MV10 clone #1 from streak library (clone #3 is wrong) | ||
* Cas-tone - | * Cas-tone - PCR-amplify CMV | ||
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** Order primers | ** Order primers | ||
* STAGE 3 - Cas-fusion | * STAGE 3 - Cas-fusion | ||
** Design primers for H2A, H2B, H3, H4, and tails | |||
** Insert Kozak-histone parts (X/N) into dCas9 (S/N) | ** Insert Kozak-histone parts (X/N) into dCas9 (S/N) | ||
* STAGE 4 - gRNA | * STAGE 4 - gRNA | ||
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'''Knock-out FLAG from N-terminus''' | '''Knock-out FLAG from N-terminus''' | ||
* | * PCR-amplify promoters | ||
** 2x 50 μL rxns | |||
** Template = CMV (KAH | |||
** f primer: XbaI-CMV f1 - 5'-CCTTTCTAGAGTTGACATTGATTATTGGCTAG | |||
** r primer: SacII-NS-CMV r1 - 5'-CATTCCGCGGGCGGCCGCTACTAGTGAGCTCTGC | |||
{| {{table}} cellspacing="3" <!-- PCR rxn table --> | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Vol | |||
| bgcolor=#cfcfcf | Mix (x2) | |||
| rowspan=7 | Expected:<br>1. CMV = 588 | |||
| rowspan=7 | [[Image:KAH042815_gel2.jpg|250px|Hover name]]<br>5 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|- | |||
| plasmid DNA || 0.5 || 1.0 | |||
|- | |||
| 10 μM XbaI-CMV f1 || 1.0 || 2.0 | |||
|- | |||
| 10 SacII-NS-CMV r1 || 1.0 || 2.0 | |||
|- | |||
| 2x GoTaq green || 25.0 || 50.0 | |||
|- | |||
| dH<sub>2</sub>O || 22.5 || 8.7 | |||
|- | |||
| || 50.0 μL || --- | |||
|} | |||
Thermal cycler: Labnet - GOTAQ | |||
* 95°C 3 min. | |||
* 30x[95°C, 30 sec; 55°C 30 sec; 72°C 30 sec] | |||
* 72°C 3 min. | |||
* 4°C, ∞ | |||
Revision as of 12:37, 11 May 2015
Karmella's BioBrick Cloning | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | |||||||||||||||||||||||
05/11/15
Cas-tone Project
Thermal cycler: Labnet - GOTAQ
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