Hosuk:LabNotes/2013-5-2: Difference between revisions

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>Hosuki78
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=====Result=====
=====Result=====
*'''S4 : RCA with Linear DNA'''
{| {{table}}
| Bright field, 20x
||
| Cy3, 20x
|-
|([[Media:CircTest_S4-LinearDNA_fig04_20x_pos02_BF.tif|Original]])
||
|([[Media:CircTest_S4-LinearDNA_fig03_20x_pos02_Cy3.tif|Original]])
|-
|[[File:CircTest_S4-LinearDNA_fig04_20x_pos02_BF.jpg|300px]]
||
|[[File:CircTest_S4-LinearDNA_fig03_20x_pos02_Cy3.jpg|300px]]
|}

Revision as of 17:01, 3 May 2013

Reverse Transcription check

  • based on Debugging plan (05/01)
Result
  • RT process seemed work.


  • +RTase sample showed very strong Cy5 signal which is supposed cDNA
  • comparing to -RTase result, the bright singal over the cell seemed they were cDNAs
  • -RTase sample has very low intensity of signals in the cell, and
  • not many spots were observed, which spots were outside of cells.
  • Most of signal came from cell structure.


  • The picture of -RTase is the position where the most number of spots were observed in all over the area.
  • In the +RTase sample, similar number or density of spots were observed in all over the area.
  • I don't know what are the large spots. Are they also cDNAs?


  • +RTase
S2:+RTase, Brighfield, 20x S2:+RTase, Cy5, 20x S2:+RTase, Merged
(Original) (Original)
File:RTCheck +RTase fig08 20xobj pos04 BF exp0.2-EM00.jpg File:RTCheck +RTase fig07 20xobj pos04 Cy5 exp0.2-EM40.jpg File:Composite +RTase fig07-08.jpg


  • -RTase (No RTase)
S3:-RTase, Brighfield, 20x S3:-RTase, Cy5, 20x S3:-RTase, Merged
(Original) (Original)
File:RTCheckNoRTase fig08 20xobj pos05 BF exp0.2-EM00.jpg File:RTCheckNoRTase fig07 20xobj pos04 Cy5 exp0.2-EM40.jpg File:Composite NoRTase fig07-08.jpg



CircLigase II reaction on the dish

  • based on Debugging plan (05/01)


  • Reaction for 2.5 hr
  • Aspirate and transfer solution from dish to tube
  • Total volume collected
    • 8~9uL from the sample at wet towel
    • about 50uL from the sample at mineral oil, but this volume include oil. After spinning the actual reaction solution seemed 8~10uL
  • Run Exo I reaction
    • 9uL of pre-circulated DNA + 1uL of Exo I
    • 6uL of wet towel sample + 1uL of Exo I
    • 6uL of mineral oil sample + 1uL of Exo I
    • 45min at 37C --> 15min at 80C


Result
  • CircLigase II reaction worked on a dish well
  • Wet towel method looked better efficiency than oil cover method did.





Rolony test with using pre-circulated DNA

  • based on Debugging plan (05/01)


  • RCA reaction for 18hour
  • Add BS(PEG)9 (20uL BS(PEG)9 + 980uL PBS)
    • S4 : Aspirate, then wash with PBS once, then add BS(PEG)9
    • S5 : Aspirate, then add BS(PEG)9
    • S6 : Aspirate, then wash with PBS once, then add BS(PEG)9
  • Block with Tris pH 8.0 : Aspirate, then wash with PBS once, then add Tris pH 8.0
  • Wash with 2x SSC once


  • Cy3-Adpater primer as usual


Result
  • S4 : RCA with Linear DNA
Bright field, 20x Cy3, 20x
(Original) (Original)
File:CircTest S4-LinearDNA fig04 20x pos02 BF.jpg File:CircTest S4-LinearDNA fig03 20x pos02 Cy3.jpg