Matt:LabNotes/2015-12-10

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Revision as of 01:00, 11 December 2015 by >Mzcai (Created page with "=CircLigase II Reagent Testing= ==Reaction== *5ul ssDNA control (10pmol total) *2ul 10X Reaction Buffer *1ul 50mM MnCl2 *4ul 5M Betaine *1ul CircLiase II Enzyme *7ul H2O *Tot...")
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CircLigase II Reagent Testing

Reaction

  • 5ul ssDNA control (10pmol total)
  • 2ul 10X Reaction Buffer
  • 1ul 50mM MnCl2
  • 4ul 5M Betaine
  • 1ul CircLiase II Enzyme
  • 7ul H2O
  • Total: 20ul

Procedure

  1. Incubated at 60C for 1hr
  2. Incubated at 80C for 10min (inactivate)
  3. Take 4ul of each and run TBU gel
  4. ssDNA Zymo column purification
    • Elute 10ul
  5. Exonuclease I Digest half of sample
    • 5ul + 1ul 10X ExoIII Buffer + 4ul H2O
    • 5ul + 1ul 10X ExoIII Buffer + 1ul Exonuclease I + 3ul H2O
  6. Incubate 37C for 45min
  7. Incubate 80C for 15min (inactivate)
  8. Run TBU gel