Hosuk:LabNotes/2013-5-20

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Revision as of 20:58, 21 May 2013 by >Hosuki78 (Created page with "====Add circular DNA (~50pmole) in fixed cells and run RCA in the oven==== *Produce circular DNA (50pmole input!) as same one made at 05/ *20uL of CircLigase II product + 30u...")
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Add circular DNA (~50pmole) in fixed cells and run RCA in the oven[edit]

  • Produce circular DNA (50pmole input!) as same one made at 05/
  • 20uL of CircLigase II product + 30uL 2x SSC in the fixed cell dish
  • Run RCA in the oven (set temp: 33C) for 19 hour
  • After RCA,
    1. Add 20 ul 250 mM BS(PEG)9 in 1 ml PBS for 1 hr at RT.
    2. Wash with PBS once
    3. Block with 1M Tris pH 8.0 for 30 min.
    4. Wash with PBS once
    5. Add 5 ul Riboshredder (Epicentre)+ 5ul RNase H(Enzymatics) in 190ul RNase H buffer(Enzymatics) for 1 hr at 37C.
    6. Rinse the entire well with ddH2O twice to remove traces of phosphate.
    7. Prepare 100uM of Cy3 labeled probe (Cy3-TCTTCAGCGTTCCCTAGA)
    8. 2uL of 100uM probes in 200uL 2x SSC
    9. Pre-heated probe solution between 60C and 80C for 5min
    10. Add to cells at room temp, and let it cool down over 5min
    11. Wash with 2x SSC (2mL, but 200uL work too) twice and image!


Result[edit]

  • RCA rolony signal were from Cy3 and FITC channel very well.
  • There were a lot of big strong spots, which were seen every time before, and I don’t think that’s rolonies,
  • they looked cluster or chuck of RCA products so the Cy3 adpaters were bound there.
  • Anyway, FITC channel represents Rolonies well…
  • Let’s try Confocal imaging!
  • Let’s move on a real protocol, from RT to RCA!


20x, Position 2[edit]

File:S4 20x pos2 052013.png


40x Oil, Position 2[edit]

File:S4 40xOil pos2 052013.png


40x Oil, Position 3[edit]

File:S4 40xOil pos3 052013.png