Matt:LabNotes/2015-12-10: Difference between revisions
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=CircLigase II Reagent Testing= | =CircLigase II Reagent Testing= | ||
*Test new CircLigase II kit that arrived this month | |||
==Reaction Mix== | ==Reaction Mix== |
Revision as of 03:20, 11 December 2015
CircLigase II Reagent Testing
- Test new CircLigase II kit that arrived this month
Reaction Mix
- 5ul ssDNA control (10pmol total)
- 2ul 10X Reaction Buffer
- 1ul 50mM MnCl2
- 4ul 5M Betaine
- 1ul CircLiase II Enzyme
- 7ul H2O
- Total: 20ul
- Sample1: New enzyme + kit reagents
- Sample2: New enzyme + Hosuk's buffer 8-1-2014 + Homemade MnCl2 & Betaine
- Sample3: Old enzyme (red cap) + kit reagents
- Sample4: No Enzyme
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
File:2015-12-10 CircLigaseII Test.jpg
Conclusion
- Both old and new enzyme work
- Circularized ssDNA actually runs slightly faster in our TBU gels