Matt:JustinLabNotes/2015-10-14

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CircLigase II Test Reagents[edit]

  • CircLigase II kit is on back order
  • I have plenty of enzyme but low on 10X buffer, 5M Betaine, and 50mM MnCl2

Reagents[edit]

Justin made from dry reagent[edit]

  • 5M Betaine
  • 50mM MnCl2

Neither worked as well as kit-provided reagents

Ordered from 3rd party companies[edit]

  • 5M Betaine from SigmaAldrich
  • 50mM MnCl2 from Trevigen


Results[edit]

File:CircLigase ii Reagents test - 10-14-15.jpg

  • Even positive control didn't circularize
  • I believe the second band is 5' adenylation from the enzyme and not circularization since in the epicentre protocol the circularized band runs slower than 70bp ladder
    • Also the circularized band showed up in Justin's gel last week
  • Try ExoI digestion to ensure