Matt:LabNotes/2014-5-27: Difference between revisions

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#*Incubate @RT for '''75 min'''
#*Incubate @RT for '''75 min'''
#**C4 & C5 Incubate @RT for '''60 min'''
#**C4 & C5 Incubate @RT for '''60 min'''
#Aspirate
#Aspirate NO WASH
#Capture with 100pM ppMALAT1
#Capture with 100pM ppMALAT1
#*Incubate @45C for 24 hours (6:30p- )
#*Incubate @45C for 24 hours (6:30p-6:30p)
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Component'''
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| Total||800
| Total||800
|}
|}
<!--#Aspirate NO WASH
#Aspirate NO WASH
#Add RCA primer: 3ul FISSEQ_ppRCA in 597 2X SSC/30%
#Add RCA primer: 4ul FISSEQ_ppRCA in 796 2X SSC/30% (1uM final concentration)
#*Pre-heat to 60C
#*Pre-heat to 60C
#*Add 100ul @45C for 15min
#*Add 100ul @45C for 15min
Line 29: Line 29:
#*Wash 0.1X SSC twice
#*Wash 0.1X SSC twice
#Add RCA mix
#Add RCA mix
#*Incubate 22hrs @ 30C
#*Incubate 21hrs @ 30C
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
|-
| H2O||513
| H2O||684
|-
|-
| 10X Phi29 Buffer||60
| 10X Phi29 Buffer||80
|-
|-
| 25mM dNTP||6
| 25mM dNTP||8
|-
|-
| 2mM aa-dUTP||12
| 2mM aa-dUTP||16
|-
|-
| Phi29||9
| Phi29||12
|-
|-
| Total||600
| Total||800
|}
|}
#Wash 1X PBS once
#Wash 1X PBS once
#12ul BS(PEG)9 in 588ul 1X PBS
#16ul BS(PEG)9 in 784ul 1X PBS
#*Incubate @RT 1hr
#*Incubate @RT 1hr
#Wash 1X PBS twice
#Wash 1X PBS twice
#Quench with 1M Tris pH 8.0 @RT 1hr
#Quench with 1M Tris pH 8.0 @RT 1hr
#*To A3-A5: 1M Tris pH 8.0
#*To D3-D5: 0.9M Tris + 50mM EDTA ph 8.0 (45mL 1M Tris + 5mL 500mM EDTA)
#Wash 1X PBS twice
#Wash 1X PBS twice
#Add 1uM dcProbe2-Cy3 in 30% formamide + 2X SSC
<!--#Add 1uM dcProbe2-Cy3 in 30% formamide + 2X SSC
#*Preheat to 75C for 5min
#*Preheat to 75C for 5min
#*Incubate @RT for 10min
#*Incubate @RT for 10min
Line 65: Line 63:


-->
-->
==Exo I/III Test==
==Exo I/III Test==


Line 71: Line 70:
**Strip with 80% formamide for 15min @RT
**Strip with 80% formamide for 15min @RT
**Wash with ddH2O twice
**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)
'''Experimental & Negative Control''' (Positions 3-12)
Line 97: Line 100:
| Total||100
| 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'''
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| 10X Exo III Buffer||20
|-
| Exo I||10
|-
| Exo III||10
|-
| H2O||160
|-
| Total||200
|}
'''Exo III: Positions 3-6'''
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| 10X Exo III Buffer||20
|-
| Exo III||10
|-
| H2O||170
|-
| Total||200
|}
'''Controls: Positions 1-2 & 11-12'''
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''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''')
===Results===
*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
***[[Matt:LabNotes/2014-5-16#Conclusion|Previous results]] show overlap is most accurate method
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Rolonies'''
| align="center" style="background:#f0f0f0;"|'''Hybridized'''
| align="center" style="background:#f0f0f0;"|'''Hybridized'''
| align="center" style="background:#f0f0f0;"|'''Hybridized'''
| align="center" style="background:#f0f0f0;"|'''Rolonies after Exo'''
| align="center" style="background:#f0f0f0;"|'''Rolonies after Exo'''
| align="center" style="background:#f0f0f0;"|'''Rolonies after Exo'''
|-
| align="center" style="background:#f0f0f0;"|'''Padlock Probe'''
| align="center" style="background:#f0f0f0;"|'''Exonuclease'''
| align="center" style="background:#f0f0f0;"|'''Position'''
| align="center" style="background:#f0f0f0;"|'''PISA'''
| align="center" style="background:#f0f0f0;"|'''bwlabel'''
| align="center" style="background:#f0f0f0;"|'''overlap'''
| align="center" style="background:#f0f0f0;"|'''PISA'''
| align="center" style="background:#f0f0f0;"|'''PISA'''
| align="center" style="background:#f0f0f0;"|'''overlap'''
| align="center" style="background:#f0f0f0;"|'''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===
*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

Latest revision as of 01:49, 31 May 2014

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