Matt:LabNotes/2014-5-27

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Artifical Rolonies

Procedure

  1. Add 140ul 100nM MALAT1_template to each well
    • Pre-heat to 85C
    • Incubate @RT for 75 min
      • C4 & C5 Incubate @RT for 60 min
  2. Aspirate NO WASH
  3. Capture with 100pM ppMALAT1
    • Incubate @45C for 24 hours (6:30p-6:30p)
Component Volume
10X Ampligase Buffer 80
2nM ppMALAT1 40
5 U/ul Ampligase 80
H2O 600
Total 800
  1. Aspirate NO WASH
  2. Add RCA primer: 4ul FISSEQ_ppRCA in 796 2X SSC/30% (1uM final concentration)
    • Pre-heat to 60C
    • Add 100ul @45C for 15min
    • Wash 2X SSC twice
    • Wash 0.1X SSC twice
  3. Add RCA mix
    • Incubate 21hrs @ 30C
Component Volume
H2O 684
10X Phi29 Buffer 80
25mM dNTP 8
2mM aa-dUTP 16
Phi29 12
Total 800
  1. Wash 1X PBS once
  2. 16ul BS(PEG)9 in 784ul 1X PBS
    • Incubate @RT 1hr
  3. Wash 1X PBS twice
  4. Quench with 1M Tris pH 8.0 @RT 1hr
  5. Wash 1X PBS twice

Exo I/III Test

Procedure

  • Use pre-made artificial MALAT1 rolonies from Matt:LabNotes/2014-5-16
    • Strip with 80% formamide for 15min @RT
    • Wash with ddH2O twice

Hybridize Padlock Probes

  • Add 100ul ppMALAT1 (dcProbe2 version) in Ampligase buffer
    • Incubate for 24 hours @ 45C

Experimental & Negative Control (Positions 3-12)

Component Volume
10X Ampligase Buffer 50
2uM ppMALAT1 2.5
H2O 447.5
Total 500

Positive Control (Positions 1-2)

Component Volume
10X Ampligase Buffer 10
H2O 90
Total 100

Digestion

  • Wash with 2X SSC once
  • Add 100ul Exonuclease mix to each well
  • Incubate @37C for 2 hrs

Exo I/III: Positions 7-10

Component Volume
10X Exo III Buffer 20
Exo I 10
Exo III 10
H2O 160
Total 200

Exo III: Positions 3-6

Component Volume
10X Exo III Buffer 20
Exo III 10
H2O 170
Total 200

Controls: Positions 1-2 & 11-12

Component Volume
10X Exo III Buffer 20
H2O 180
Total 200

Detection

  • Wash with 2X SSC once
  • Add 100ul 1uM dcProbe2RevComp in 30% formamide + 2X SSC for 10min @ RT
    • Preheated to 75C
  • Aspirate ALL
  • Wash with 2X SSC twice
  • Image (Saved in 5-28-2014)

Check Digestion of Rolonies

  • Add 80% formamide in 2X SSC for 15min @37C
    • Preheated to 75C
  • Wash with ddH2O twice
  • Add 100ul 1uM dcProbe1-Cy3 in 30% formamide + 2X SSC for 10min @ RT to Positions 3-12
    • Preheated to 75C
  • Aspirate ALL
  • Wash with 2X SSC twice
  • Image (Saved in 5-30-2014)