Matt:LabNotes/2014-5-27: Difference between revisions
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#Aspirate NO WASH | #Aspirate NO WASH | ||
#Add RCA primer: 4ul FISSEQ_ppRCA in 796 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 | ||
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#*Wash 0.1X SSC twice | #*Wash 0.1X SSC twice | ||
#Add RCA mix | #Add RCA mix | ||
#*Incubate 20hrs @ 30C | #*Incubate 20hrs @ 30C (7p- ) | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
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===Digestion=== | ====Digestion==== | ||
*Wash with 2X SSC once | *Wash with 2X SSC once | ||
*Add 100ul Exonuclease mix to each well | *Add 100ul Exonuclease mix to each well |
Revision as of 02:22, 29 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 20hrs @ 30C (7p- )
Component | Volume |
H2O | 684 |
10X Phi29 Buffer | 80 |
25mM dNTP | 8 |
2mM aa-dUTP | 16 |
Phi29 | 12 |
Total | 800 |
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)