Hosuk:LabNotes/2013-4-24: Difference between revisions

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>Hosuki78
(Created page with "====RCA with Pre-circularized DNA on fixed cells==== =====Procedure===== *Run 18hr. RCA with using CircLigase II product got from 4/24 result **7uL of product + 93uL 2x SSC ...")
 
>Hosuki78
Line 22: Line 22:
*Are there problem in washing step? or BS(PEG)9 procedure?  
*Are there problem in washing step? or BS(PEG)9 procedure?  
*Need to ask to Jay
*Need to ask to Jay
{| {{table}}
| 20x Obj. Bright field
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| 20x Obj. Cy3
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| Merged
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|([[Media:S1_washPBS3times_PreCirc_RCA18hr_Cy3Adapt_20x_pos02_fig04_BF_Exp0.2-EM00.tif|Original]])
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|([[Media:S1_washPBS3times_PreCirc_RCA18hr_Cy3Adapt_20x_pos02_fig03_Cy3_Exp0.2-EM40.tif|Original]])
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|[[File:S1_washPBS3times_PreCirc_RCA18hr_Cy3Adapt_20x_pos02_fig04_BF_Exp0.2-EM00.jpg|300px]]
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|[[File:S1_washPBS3times_PreCirc_RCA18hr_Cy3Adapt_20x_pos02_fig03_Cy3_Exp0.2-EM40.jpg|300px]]
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|[[File:Composite_fig03-04.jpg|300px]]
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Revision as of 16:59, 25 April 2013

RCA with Pre-circularized DNA on fixed cells

Procedure
  • Run 18hr. RCA with using CircLigase II product got from 4/24 result
    • 7uL of product + 93uL 2x SSC in the fixed cell dish
    • incubate 15min at RT
    • Wash with PBS once
    • Run RCA with the same step as rolony protocol
      • 1uL of 100uM RCA primer in 199uL 2x SSC
      • Add to cell, incubate for 15min at 60C
      • Aspirate, and wash using 2x SSC twice
      • Aspirate, and wash using 0.1x SSC twice
      • Add RCA mix
    • 18hr in 30C Oven


Result
  • FL spots looked similar number and similar density to what I’ve got before.
  • Those seemed rolony but not many.
  • It is suspected that rolonies didn't bind inside cell so they could be washed away.
  • Are there problem in washing step? or BS(PEG)9 procedure?
  • Need to ask to Jay


20x Obj. Bright field 20x Obj. Cy3 Merged
(Original) (Original)
File:S1 washPBS3times PreCirc RCA18hr Cy3Adapt 20x pos02 fig04 BF Exp0.2-EM00.jpg File:S1 washPBS3times PreCirc RCA18hr Cy3Adapt 20x pos02 fig03 Cy3 Exp0.2-EM40.jpg File:Composite fig03-04.jpg