Matt:LabNotes/2014-5-13: Difference between revisions

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#Quench with 1M Tris pH 8.0 @RT for 30min
#Quench with 1M Tris pH 8.0 @RT for 30min
#Wash with 1X PBS twice
#Wash with 1X PBS twice
 
====Detection====
#Add 0.5uM dcProbe1-Cy3 in 30% formamide + 2X SSC
#Add 0.5uM dcProbe1-Cy3 in 30% formamide + 2X SSC
#*Preheat to 75C for 5min
#*Preheat to 75C for 5min
Line 60: Line 60:
#Wash with 2X SSC twice
#Wash with 2X SSC twice
#Image with Confocal
#Image with Confocal
#*Laser 3% & Gain 550
#Strip with 80% formamide for 10min @RT
#*Preheated to 75C
#Wash with H2O twice


===Results===
===Results===

Revision as of 23:41, 16 May 2014

MALAT1 Rolonies

Procedure

  1. After fixing with 10% formalin 15min 37C & then sitting in 70% EtOH, Wash twice with 1X PBS
  2. Add 140ul 100nM MALAT1_template to wells C2 & B4
    • Pre-heat to 85C
    • Incubate @RT for 15 min
  3. Aspirate & Wash once with 1X PBS
  4. Capture with 100pM ppMALAT1_dcProbe1
    • Incubate @45C for 24 hours
Component Volume
10X Ampligase Buffer 20
1nM ppMALAT1_dcProbe1 20
5 U/ul Ampligase 20
H2O 140
Total 200
  1. Aspirate NO WASH
  2. Add RCA primer: 1ul FISSEQ_ppRCA in 199ul 2X SSC/30%
    • Pre-heat to 60C
    • Add 100ul @45C for 15min
    • Wash 2X SSC twice
    • Wash 0.1X SSC twice
  3. Add RCA mix
    • Incubate 22hrs @ 30C
Component Volume
H2O 171
10X Phi29 Buffer 20
25mM dNTP 2
2mM aa-dUTP 4
Phi29 3
Total 200
  1. Wash with 1X PBS once
  2. 4ul BS(PEG)9 in 196ul 1X PBS
    • Incubate 1hr @ RT
  3. Wash with 1X PBS twice
  4. Quench with 1M Tris pH 8.0 @RT for 30min
  5. Wash with 1X PBS twice

Detection

  1. Add 0.5uM dcProbe1-Cy3 in 30% formamide + 2X SSC
    • Preheat to 75C for 5min
    • Incubate @RT for 10min
  2. Wash with 2X SSC twice
  3. Image with Confocal
    • Laser 3% & Gain 550
  4. Strip with 80% formamide for 10min @RT
    • Preheated to 75C
  5. Wash with H2O twice

Results