Revision as of 18:26, 30 May 2014 by >Mzcai
Artifical Rolonies
Procedure
- 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
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
- 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
- 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)