Hosuk:LabNotes/2014-8-19: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Hosuki78
>Hosuki78
Line 90: Line 90:


=====Padlock Probe capture, 2nd Rolony Process=====
=====Padlock Probe capture, 2nd Rolony Process=====
#Process
#Strip Cy3-1st Rolony detection Probes with preheated 80% formamide at 70C, incubate for 15min
*#Strip Cy3-1st Rolony detection Probes with preheated 80% formamide at 70C, incubate for 15min
*#Wash with PBS 3 times
*#Wash with PBS 3 times
*#Prepare Agi26k0gap : 50nM in 120uL (Matt's product) --> 30uL in 100uL 2x SSC, which is 15nM
*#Prepare Agi26k0gap : 50nM in 120uL (Matt's product) --> 30uL in 100uL 2x SSC, which is 15nM
Line 111: Line 110:
*#Wash with PBS twice, add 1M Tris pH8.0 , incubate at room temp. for 1hr.
*#Wash with PBS twice, add 1M Tris pH8.0 , incubate at room temp. for 1hr.
*#Wash with PBS twice, store at PBS
*#Wash with PBS twice, store at PBS


====Decoding Procedure====
====Decoding Procedure====
*
*
*#dcProbe set
*#dcProbe set

Revision as of 20:24, 22 August 2014


Decoding with Agi26k0gap Padlock Probe

  • Human brain tissue from Yun
    • HB3, section 9
    • Total 15 tissue sections
  • Mouse tissue : Total 15 tissue sections
  • Stored in -80C


Procedure

Fixation
  • Process
    1. 4% Formaldehyde : 2.5mL 16% Paraformaldehyde + 1mL 10x DEPC-PBS + 6.5mL DEPC-H2O (Tot. 10mL)
      • 16% Paraformaldehyde Solution)
      • Manufacture : Electron Microscopy Diatome --> 16% PARAFORMALDEHYDE AQ SOLUTN 50-980-486, No.:15700
      • Supply : (Fisher Scientific), 50980486
      • Paraformaldehyde - methanol free solution. A more efficient and rapid penetrant fixative used in combination with Glutaraldehyde and Acrolein fixatives. Easy to break, prescored, 10 ml ampoules sealed under inert gas 10 ml
    2. Take Tissue glass slide from -80C, and put them to room temp. for 2min.
    3. Add 4% formamide on a tissue, incubate for 30min at room temp.
    4. Wash with PBS 3 times (add PBS in 3 wells of 6 well plate and series washing with glasses)
    5. Attach glass with glue
      • Cyanoacrylate adhesives : High-viscosity, Sigma-Aldrich, Z105902-1EA
    6. Wipe PBS from the glass (around tissue)
    7. Apply glue along the edge of the glass, put on the bottom of MatTek dish, wait for 5min

File:TissueGlassOnMatTek 2.png File:TissueGlassOnMatTek 1.png

1st Rolony Process
  • RT
    1. Wash with H2O for 5min. twice
    2. Add 0.25% TX-100 + DEPC-H2O, incubate for 15min. at room temp.
    3. Wash with DEPC-H2O twice
    4. Add 200 ul 0.1% pepsin in 0.1 N HCl (4uL of 5% Pepsin + 10uL of 2N HCl + 186uL DEPC-H2O), incubate for 10 min at 37C
    5. Wash with DEPC-PBS three times to inactivate pepsin
    6. Add RT mix (Hexamer, -a-dUTP)
      • DEPC-H2O 161uL
      • M-MuLV Buffer 20uL
      • 25mM dNTP 2uL
      • 100uM random hexamer RT Primer 5uL
      • Rnase Inhibitor 2uL
      • M-MuLV reverse transcriptase 10 uL
    7. Incubate the specimen with the reaction mixture for 10 min at 4°C, then transfer to 37°C for overnight.


  • Circligase II
    1. Wash with PBS once, skip BS(PEG)9
    2. Add RNase H mix, incubate for 1hr. at 37C
      • H2O 168uL
      • Riboshredder 2uL
      • RNase H buffer 20uL
      • RNase H 10uL
    3. Wash with nucleaus-free H2O twice
    4. Add CircLigase II mix, incubate at 60C for 2hr.
      • H2O 128uL
      • Buffer 20uL
      • 50mM MnCl2 10uL
      • 5M Betaine 40uL
      • CircLigaseII 2uL


  • RCA
    1. Add 0.5uM RCA Primer in 200uL (2x SSC + 30% Formamide), incubate at 60C for 1hr.
      • 1uL of 100uM RCA Primer + 199uL (2X SSC + 30% Formamide)
    2. Wash with 2x SSC, 1x SSC twice each
    3. Prepare RCA reaction mix
      • H2O 172uL
      • Buffer 20uL
      • 25mM dNTP 2uL
      • 2mM a-dUTP 4uL
      • Phi29(100 U/µl) 2uL
    4. Add RCA mix, incubate at 30C for overnight


  • Post fix
    1. Wash with PBS once
    2. add 200uL BS(PEG)9 mix (4uL BS(PEG)9 in 196uL 1x PBS), incubate for 1hr at RT
    3. Wash with PBS once, add Tris pH 8.0, incubate for 30min
    4. Wash with PBS once


  • Imaging
    1. Prepare detection Probe : Final Conc. 1uM --> 2uL of 100uM Cy3-Adapt + 198uL (2xSSC + 30% Formamide)
    2. Preheat detection Probe at 80C for 5min
    3. Add to sample and incubate at RT for 10min
    4. Wash two times for 1 minute each with 1 mL 1X PBS


Padlock Probe capture, 2nd Rolony Process
  1. Strip Cy3-1st Rolony detection Probes with preheated 80% formamide at 70C, incubate for 15min
    1. Wash with PBS 3 times
    2. Prepare Agi26k0gap : 50nM in 120uL (Matt's product) --> 30uL in 100uL 2x SSC, which is 15nM
      • 30uL Agi26k0gap (15nM)
      • 10uL Ampligase (5 U/uL)
      • 10uL Buffer
      • 50uL H2O
    3. Add 100uL per MatTek dish, incubate dishes at 37C for 30min, and 45C for overnight
    4. Wash with PBS twice,
    5. Preheat 0.5uM FISSEQ_ppRCA primer in 2X SSC at 70C, and add and incubate it at 45C for 30min
    6. Wash with PBS twice
    7. Add RCA mix, incubate at 30C for overnight (start : 08/12 3pm)
      • H2O : 172uL
      • 10x Buffer : 20uL
      • dNTP : 2uL
      • a-dUTP : 4uL
      • Phi29 (100 U/µl) : 2uL
    8. Wash with PBS once, add BS(PEG)9, incubate at room temp. for 1hr.
    9. Wash with PBS twice, add 1M Tris pH8.0 , incubate at room temp. for 1hr.
    10. Wash with PBS twice, store at PBS

Decoding Procedure

    1. dcProbe set