Matt:LabNotes/2015-12-10
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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
- Sample1: All kit provided reagents
- Sample2: Hosuk's buffer 8-1-2014
- Sample3: Use my MnCl2
- Sample4: Use my Betaine
Procedure
- Incubated at 60C for 1hr
- Incubated at 80C for 10min (inactivate)
- Take 4ul of each and run TBU gel
- ssDNA Zymo column purification
- Elute 10ul
- Exonuclease I Digest half of sample
- 5ul + 1ul 10X ExoIII Buffer + 4ul H2O
- 5ul + 1ul 10X ExoIII Buffer + 1ul Exonuclease I + 3ul H2O
- Incubate 37C for 45min
- Incubate 80C for 15min (inactivate)
- Run TBU gel