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
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

  1. After fixing with 10% formalin 15min 37C & then sitting in 70% EtOH, Wash twice with 1X PBS
  2. Add 140ul 100nM MALAT1_template to wells C2 & B4
    • Pre-heat to 85C
    • Incubate @RT for 15 min
  3. Aspirate & Wash once with 1X PBS
  4. 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
  1. Aspirate NO WASH
  2. 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
  3. 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