User:Cassandra M Barrett/Notebook/Open Chromatin/2015/09/29

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Preparing parts for LCR

Purpose: digest MV10 and blunt the cut ends for LCR

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 1ul MBN once tube is cooled. Incubate at RT 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.

Quantify resulting DNA: 40ng/uL

Gel ran weird and wasn't able to isolate a band...extracted any way. Band was very bright.



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