Hosuk:LabNotes/2013-4-22

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Next Plan[edit]

RCA test[edit]
  • Run RCA reaction in a tube with CircLigase II reaction sample using custom DNA
  • Since the custom DNA has the sequence for binding to RCA primer,
  • I could see the signal from RCA product.


Experiment plan for RCA test[edit]
    • Run CircLigase II with custom ssDNA (2hr. @ 60C)
    • Run Exo I (1hr. @ 37C) (--> Gel running)
    • Run RCA
      • Tube1 : custom ssDNA + RCA (18hr. @ 30C)
      • Tube2 : Circ + RCA (18hr. @ 30C)
      • Tube3 : Circ and Exo I + RCA (18hr. @ 30C)

File:RCAReationsinTube.jpg


Jay's advice[edit]
  • (As following Jay's suggestion, 18hour RCA was done from yesterday to today using fixed cells, but couldn't get any reasonable result. There were some FL signal, but it was false signal)
    • Jay's suggestion
      • for now, just wash the cells well before you fix. (2 ml PBS x 3)
      • after fixing cells and treating with etoh, wash with PBS.
      • then just dilute your DNA in 200 ul of 2x SSC and incubate at RT for 15 min.
      • wash with 2xSSC several times.
      • then just do your RCA. (with aminoallyl dUTP), followed by the BS(PEG)9 step. then hybridize. You should get a lot of rolonies.


CircLigase II test to prove what is circularized DNA[edit]

Sample set[edit]
  • Run CircLigase II reaction
    • input ssDNA : Custom ssDNA (50bp) having adapter sequence
    • +CircLigaseII and -CircLigase II
    • Reaction condition : 60C Thermo Cycler for 1hr 30min. --> 80C for 10min
Reaction condition[edit]
  • CircLigase II : 20uL total reaction volume
Reagent Conc. Volume Final Conc.
ss DNA 2uM 5uL 0.5uM
10x Reaction Buffer 10x 2uL 1x
MnCl2 50mM 1uL 2.5mM
Betain 5M 4uL 1M
CircLigase II 100U/uL 1uL 5U/uL
H2O 7uL


  • Run Exo I reaction
    • 9uL from CircLigase and CircLigase II reaction
    • add 1uL of Exo I
    • Run 37C for 1hr. --> 80C for 15min


Result[edit]
  • TBU gel (All samples were denatured by 75C for 8min)
  • Result
    • -CircLigase II showed the same band to Custom ssDNA,
    • and Exo I reaction of –CircLigase II showed nothing (Lane 7).
    • Thus, the band in +CircLigase II and Exo I reaction of +CircLigase II are circularized DNA.
    • In result, circularized 50bp of custom ssDNA (CircAdapt2 Primer) runs faster than linear one.
    • This results are consistent with the previous results.
    • RCA reaction is running with two samples, +CircLigase II and Exo I of +CircLigase II.


  • Gel table
#1 #3 #4 #5 #6 #7
10bp Custom ssDNA +CircLigase II Exo I of +CircLigase II -CircLigase II Exo I of -CircLigase II