Matt:LabNotes/2015-12-10: Difference between revisions
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>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...") |
>Mzcai m (→Reaction) |
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Line 1: | Line 1: | ||
=CircLigase II Reagent Testing= | =CircLigase II Reagent Testing= | ||
==Reaction== | ==Reaction Mix== | ||
*5ul ssDNA control (10pmol total) | *5ul ssDNA control (10pmol total) | ||
*2ul 10X Reaction Buffer | *2ul 10X Reaction Buffer | ||
Line 10: | Line 10: | ||
*Total: 20ul | *Total: 20ul | ||
*Sample1: | *Sample1: New enzyme + kit reagents | ||
*Sample2: [[Hosuk:LabNotes/2014-3-24 | Hosuk's buffer 8-1-2014]] | *Sample2: New enzyme + [[Hosuk:LabNotes/2014-3-24 | Hosuk's buffer 8-1-2014]] + Homemade MnCl2 & Betaine | ||
*Sample3: | *Sample3: Old enzyme (red cap) + kit reagents | ||
*Sample4: | *Sample4: No Enzyme | ||
===Procedure=== | ===Procedure=== | ||
Line 27: | Line 27: | ||
#Incubate 80C for 15min (inactivate) | #Incubate 80C for 15min (inactivate) | ||
#Run TBU gel | #Run TBU gel | ||
[[File:2015-12-10_CircLigaseII_Test.jpg|650px]] | |||
==Conclusion== | |||
*Both old and new enzyme work | |||
*Circularized ssDNA actually runs slightly faster in our TBU gels |
Revision as of 03:19, 11 December 2015
CircLigase II Reagent Testing
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