User:Cassandra M Barrett/Notebook/Open Chromatin/2015/10/03
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Cutting and Blunting MV10
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.
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.
The gel shows a large bright band on the cut lane between the relaxed and supercoiled plasmid lanes on the MV10 uncut control. The middle band is the only one present on the cut lane indicating effectiveness of the enzyme. As compared to the first time I ran a gel like this, the cutting is much much more effective. The bright band was extracted and eluted in warm elution solution to yield a concentration of 81ng/uL!