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''' | ||
Line 22: | Line 22: | ||
| Total||800 | | Total||800 | ||
|} | |} | ||
#Aspirate NO WASH | |||
#Add RCA primer: | #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 | #*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|| | | H2O||684 | ||
|- | |- | ||
| 10X Phi29 Buffer|| | | 10X Phi29 Buffer||80 | ||
|- | |- | ||
| 25mM dNTP|| | | 25mM dNTP||8 | ||
|- | |- | ||
| 2mM aa-dUTP|| | | 2mM aa-dUTP||16 | ||
|- | |- | ||
| Phi29|| | | Phi29||12 | ||
|- | |- | ||
| Total|| | | Total||800 | ||
|} | |} | ||
#Wash 1X PBS once | #Wash 1X PBS once | ||
# | #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 | ||
#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]
- Add 140ul 100nM MALAT1_template to each well
- Pre-heat to 85C
- Incubate @RT for 75 min
- C4 & C5 Incubate @RT for 60 min
- Aspirate NO WASH
- 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 |
- Aspirate NO WASH
- 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
- 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 |
- Wash 1X PBS once
- 16ul BS(PEG)9 in 784ul 1X PBS
- Incubate @RT 1hr
- Wash 1X PBS twice
- Quench with 1M Tris pH 8.0 @RT 1hr
- 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
- Previous results show overlap is most accurate method
' | ' | ' | 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