Revision as of 01:49, 31 May 2014 by >Mzcai
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
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
- 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
- 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
- Wash with ddH2O twice
- Add 100ul 1uM dcProbe1-Cy3 in 30% formamide + 2X SSC for 10min @ RT to Positions 3-12
- 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