Matt:LabNotes/2015-12-10: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>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
 
(2 intermediate revisions by the same user not shown)
Line 1: Line 1:
=CircLigase II Reagent Testing=
=CircLigase II Reagent Testing=
*Test new CircLigase II kit that arrived this month


==Reaction==
==Reaction Mix==
*5ul ssDNA control (10pmol total)
*5ul ssDNA control (10pmol total)
*2ul 10X Reaction Buffer
*2ul 10X Reaction Buffer
Line 10: Line 11:
*Total: 20ul
*Total: 20ul


*Sample1: All kit provided reagents
*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: Use my MnCl2
*Sample3: Old enzyme (red cap) + kit reagents
*Sample4: Use my Betaine
*Sample4: No Enzyme


===Procedure===
===Procedure===
Line 27: Line 28:
#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
*Our homemade components work just as well
*Circularized ssDNA actually runs slightly faster in our TBU gels

Latest revision as of 22:32, 14 December 2015

CircLigase II Reagent Testing[edit]

  • Test new CircLigase II kit that arrived this month

Reaction Mix[edit]

  • 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[edit]

  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

File:2015-12-10 CircLigaseII Test.jpg

Conclusion[edit]

  • Both old and new enzyme work
  • Our homemade components work just as well
  • Circularized ssDNA actually runs slightly faster in our TBU gels