Matt:LabNotes/2014-5-13: Difference between revisions
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>Mzcai (Created page with "==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 ...") |
>Mzcai m (→Procedure) |
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Line 20: | Line 20: | ||
|- | |- | ||
| H2O||140 | | 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 (5:30pm- | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| H2O||171 | |||
|- | |||
| 10X Phi29 Buffer||20 | |||
|- | |||
| 25mM dNTP||2 | |||
|- | |||
| 2mM aa-dUTP||4 | |||
|- | |||
| Phi29||3 | |||
|- | |- | ||
| Total||200 | | Total||200 | ||
|} | |} |
Revision as of 21:56, 14 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 (5:30pm-
Component | Volume |
H2O | 171 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
2mM aa-dUTP | 4 |
Phi29 | 3 |
Total | 200 |