Matt:LabNotes/2014-5-13: Difference between revisions
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>Mzcai m (→Procedure) |
>Mzcai mNo edit summary |
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#*Wash 0.1X SSC twice | #*Wash 0.1X SSC twice | ||
#Add RCA mix | #Add RCA mix | ||
#*Incubate 22hrs @ 30C | #*Incubate 22hrs @ 30C | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
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| Total||200 | | Total||200 | ||
|} | |} | ||
#Wash with 1X PBS once | |||
#4ul BS(PEG)9 in 196ul 1X PBS | |||
#*Incubate 1hr @ RT | |||
#Wash with 1X PBS twice | |||
#Quench with 1M Tris pH 8.0 @RT for 30min |
Revision as of 20:20, 15 May 2014
MALAT1 Rolonies
Procedure
- After fixing with 10% formalin 15min 37C & then sitting in 70% EtOH, Wash twice with 1X PBS
- Add 140ul 100nM MALAT1_template to wells C2 & B4
- Pre-heat to 85C
- Incubate @RT for 15 min
- Aspirate & Wash once with 1X PBS
- Capture with 100pM ppMALAT1
- Incubate @45C for 24 hours
Component | Volume |
10X Ampligase Buffer | 20 |
1nM ppMALAT1 | 20 |
5 U/ul Ampligase | 20 |
H2O | 140 |
Total | 200 |
- Aspirate NO WASH
- Add RCA primer: 1ul FISSEQ_ppRCA in 199ul 2X SSC/30%
- Pre-heat to 60C
- Add 100ul @45C for 15min
- Wash 2X SSC twice
- Wash 0.1X SSC twice
- Add RCA mix
- Incubate 22hrs @ 30C
Component | Volume |
H2O | 171 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
2mM aa-dUTP | 4 |
Phi29 | 3 |
Total | 200 |
- Wash with 1X PBS once
- 4ul BS(PEG)9 in 196ul 1X PBS
- Incubate 1hr @ RT
- Wash with 1X PBS twice
- Quench with 1M Tris pH 8.0 @RT for 30min