Matt:LabNotes/2014-5-27

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Revision as of 01:49, 31 May 2014 by >Mzcai (→‎Exo I/III Test)
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Artifical Rolonies[edit]

Procedure[edit]

  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[edit]

Procedure[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

  • 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)

Results[edit]

  • Counted using 3 methods:
    • PISA: looks at gradient of intensity (can overcount if lots of noise)
    • bwlabel: counts features after setting a threshold of 9000 (can undercount)
    • overlap: uses PISA but then only counts ones that also overlap with aligned rolony image
' ' ' Rolonies Hybridized Hybridized Hybridized Rolonies after Exo Rolonies after Exo Rolonies after Exo
Padlock Probe Exonuclease Position PISA bwlabel overlap PISA PISA overlap bwlabel
-ppMALAT1 -Exo 1 930 2 4 85 N/A N/A N/A
-ppMALAT1 -Exo 2 1562 3 2 16 N/A N/A N/A
+ppMALAT1 Exo III 3 3134 3 34 286 427 405 349
+ppMALAT1 Exo III 4 4352 5 17 89 591 545 495
+ppMALAT1 Exo III 5 4997 2 55 206 2441 1888 2542
+ppMALAT1 Exo III 6 9189 9 4 18 4715 3687 4714
+ppMALAT1 Exo I/III 7 1272 3 0 26 0 0 2
+ppMALAT1 Exo I/III 8 1062 1 3 109 0 0 3
+ppMALAT1 Exo I/III 9 3480 2 10 78 0 0 10
+ppMALAT1 Exo I/III 10 3758 3 2 16 0 0 13
+ppMALAT1 -Exo 11 3315 1880 2209 3335 5866 2825 7620
+ppMALAT1 -Exo 12 1909 992 1414 3056 3521 1645 4543

Conclusion[edit]

  • Clearly shows Exo III and Exo I/III are enough to digest padlock probes and prevent them from hybridizing to rolonies
    • Exo I does a more complete digestion as evidenced by lower counts
    • Control (Pos11-12) shows about 70% rolonies detected by hybridized padlock probes
  • Digestion of rolonies
    • Control (Pos11-12) with no Exonuclease showed 85% of rolonies counted with overlap count
    • Exo III showed 13%-40% of rolonies counted
    • Exo I/III showed 0% of rolonies counted
  • Therefore estimate ~60% of rolonies were digested by Exo III