Matt:LabNotes/2014-5-27: Difference between revisions

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>Mzcai
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#*Incubate @RT for '''75 min'''
#*Incubate @RT for '''75 min'''
#**C4 & C5 Incubate @RT for '''60 min'''
#**C4 & C5 Incubate @RT for '''60 min'''
#Aspirate
#Aspirate NO WASH
#Capture with 100pM ppMALAT1
#Capture with 100pM ppMALAT1
#*Incubate @45C for 24 hours (6:30p- )
#*Incubate @45C for 24 hours (6:30p-6:30p)
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Component'''
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| Total||800
| Total||800
|}
|}
<!--#Aspirate NO WASH
#Aspirate NO WASH
#Add RCA primer: 3ul FISSEQ_ppRCA in 597 2X SSC/30%
#Add RCA primer: 4ul FISSEQ_ppRCA in 796 2X SSC/30%
#*Pre-heat to 60C
#*Pre-heat to 60C
#*Add 100ul @45C for 15min
#*Add 100ul @45C for 15min
Line 29: Line 29:
#*Wash 0.1X SSC twice
#*Wash 0.1X SSC twice
#Add RCA mix
#Add RCA mix
#*Incubate 22hrs @ 30C
#*Incubate 20hrs @ 30C
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
|-
| H2O||513
| H2O||684
|-
|-
| 10X Phi29 Buffer||60
| 10X Phi29 Buffer||80
|-
|-
| 25mM dNTP||6
| 25mM dNTP||8
|-
|-
| 2mM aa-dUTP||12
| 2mM aa-dUTP||16
|-
|-
| Phi29||9
| Phi29||12
|-
|-
| Total||600
| Total||800
|}
|}
#Wash 1X PBS once
<!--#Wash 1X PBS once
#12ul BS(PEG)9 in 588ul 1X PBS
#16ul BS(PEG)9 in 784ul 1X PBS
#*Incubate @RT 1hr
#*Incubate @RT 1hr
#Wash 1X PBS twice
#Wash 1X PBS twice
Line 71: Line 71:
**Strip with 80% formamide for 15min @RT
**Strip with 80% formamide for 15min @RT
**Wash with ddH2O twice
**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)
'''Experimental & Negative Control''' (Positions 3-12)
Line 97: Line 101:
| Total||100
| 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'''
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| 10X Exo III Buffer||20
|-
| Exo I||10
|-
| Exo III||10
|-
| H2O||160
|-
| Total||200
|}
'''Exo III: Positions 3-6'''
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| 10X Exo III Buffer||20
|-
| Exo III||10
|-
| H2O||170
|-
| Total||200
|}
'''Controls: Positions 1-2 & 11-12'''
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''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''')

Revision as of 00:55, 29 May 2014

Artifical Rolonies

Procedure

  1. Add 140ul 100nM MALAT1_template to each well
    • Pre-heat to 85C
    • Incubate @RT for 75 min
      • C4 & C5 Incubate @RT for 60 min
  2. Aspirate NO WASH
  3. 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
  1. Aspirate NO WASH
  2. Add RCA primer: 4ul FISSEQ_ppRCA in 796 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 20hrs @ 30C
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)