User:Cassandra M Barrett/Notebook/Open Chromatin/2015/10/03: Difference between revisions
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== | ==Cutting and Blunting MV10== | ||
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Purpose: digest MV10 and blunt the cut ends for LCR. I decided I wasn't happy with not being able to band isolate. I'll run the LCR with the MV10 I prepped on 10/1 as well as this version, which will be more carefully extracted. | |||
Methods: | |||
Digest MV10 (concentration: 266ng/uL) with XbaI | |||
*MV10 plasmid 15uL | |||
*XbaI 1uL | |||
*Cutsmart Buffer 2uL | |||
*Water 2uL | |||
Incubate at 37C for 15min (fast acting enzyme). Heat inactivate at 65C for 5min. Cool to RT for 10min. Add 2.6ul MBN once tube is cooled. Incubate at 30C for 30min. Run entire sample on gel (1% agrose, 45 min, 110V) and clean up band that correlates with cut plasmid (Middle band, run with lane of uncut MV10- 1uL- as a control). Use Sigma GenElute gel extraction kit. | |||
Results: | |||
Revision as of 09:35, 3 October 2015
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Cutting and Blunting MV10Purpose: digest MV10 and blunt the cut ends for LCR. I decided I wasn't happy with not being able to band isolate. I'll run the LCR with the MV10 I prepped on 10/1 as well as this version, which will be more carefully extracted. Methods: Digest MV10 (concentration: 266ng/uL) with XbaI
Incubate at 37C for 15min (fast acting enzyme). Heat inactivate at 65C for 5min. Cool to RT for 10min. Add 2.6ul MBN once tube is cooled. Incubate at 30C for 30min. Run entire sample on gel (1% agrose, 45 min, 110V) and clean up band that correlates with cut plasmid (Middle band, run with lane of uncut MV10- 1uL- as a control). Use Sigma GenElute gel extraction kit.
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