Matt:LabNotes/2014-5-27: Difference between revisions
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>Mzcai m (→Exo I/III Test) |
>Mzcai |
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**Preheated to 75C | **Preheated to 75C | ||
*Wash with ddH2O twice | *Wash with ddH2O twice | ||
*Add 100ul 1uM dcProbe1-Cy3 in 30% formamide + 2X SSC for 10min @ RT | *Add 100ul 1uM dcProbe1-Cy3 in 30% formamide + 2X SSC for 10min @ RT to Positions 3-12 | ||
**Preheated to 75C | **Preheated to 75C | ||
*Aspirate ALL | *Aspirate ALL | ||
*Wash with 2X SSC twice | *Wash with 2X SSC twice | ||
*Image (Saved in '''5- | *Image (Saved in '''5-30-2014''') |
Revision as of 18:26, 30 May 2014
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
- 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
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
- 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)