Hosuk:LabNotes/2013-4-23

From ZhangLabWiki
Jump to navigation Jump to search

RCA test with the product of CircLigase II reaction[edit]

Goal[edit]
  • Verify that there are circularized DNA in the CircLigase II reaction sample


Experiment Procedure[edit]
  • Run RCA
    • S1 : CircLigase II reaction sample (18hr. @ 30°C)
    • S2 : -CircLigase II sample (No CircLigase II Enzyme while CircLigase II reaction) (18hr. @ 30°C)
    • Protocol
    • File:RCAReationProtocolinTube.jpg


  • Mixing Cy3-Adapter
    • 4.5uL of each RCA sample + 0.5uL of 100uM Cy3-Adapter
    • S1 : +CircLigase II + Cy3-Adapter
    • S2 : -CircLigase II + Cy3-Adapter
    • S3 : H2O + Cy3-Adapter
    • small drop(0.6uL) of each sample on a glass slide and cover with cover slide, and imaging
Result[edit]
  • According to the image of RCA with +CircLigase II, there are RCA product.
  • So, CircLigase II reaction was done.
  • Again, circularized 50bp DNA ran faster than linear in the 20% TBU gel.
S1:RCA, +CircLigase S2:RCA, -CircLigase S3:H2O
(Original) (Original) (Original)
File:CircII Custom RCA Cy3Adapter 20x fig02.jpg File:-CircII(noenzyme) Custom RCA Cy3Adapter 20x fig02.jpg File:H2O Cy3Adapter 20x fig02.jpg


Next Plan[edit]
  • Test in-situ RCA using circularized DNA into fixed cell
    • Add circularized DNA into fixed cell, and incubte for 15min at RT
    • Wash 2xSSC several times (maybe twice)
    • Run RCA (including aminoallyl dUTP)
    • Priming Cy3-Adapter and image the Rolonies