Matt:LabNotes/2014-5-7: Difference between revisions

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>Mzcai
(Created page with "==ppMALAT1_dcProbe1 Capture of MALAT1 Rolonies +/- EDTA== ===Procedure=== *Add 100ul ppMALAT1_dcProbe1 with 0.5 U/ul Ampligase **A3&D3: 10pM **A4&D4: 50pM **A5&D5: 2uM **Incu...")
 
>Mzcai
mNo edit summary
Line 87: Line 87:
*Wash 2X SSC twice
*Wash 2X SSC twice
*Image
*Image
**Used Pinhole: 2.0
===Results===
===Results===
*Saved in 5-8-2014
*Saved in 5-8-2014

Revision as of 22:53, 8 May 2014

ppMALAT1_dcProbe1 Capture of MALAT1 Rolonies +/- EDTA

Procedure

  • Add 100ul ppMALAT1_dcProbe1 with 0.5 U/ul Ampligase
    • A3&D3: 10pM
    • A4&D4: 50pM
    • A5&D5: 2uM
    • Incubate @45C for 24 hours

10pM

Component Volume
10X Ampligase Buffer 20
5 U/ul Ampligase 20
1nM ppMALAT1_dcProbe1 2
H2O 158
Total 200

50pM

Component Volume
10X Ampligase Buffer 20
5 U/ul Ampligase 20
1nM ppMALAT1_dcProbe1 10
H2O 150
Total 200

2uM

Component Volume
10X Ampligase Buffer 20
5 U/ul Ampligase 20
100uM ppMALAT1_dcProbe1 4
H2O 156
Total 200
  • Aspirate NO WASH
  • Add RCA primer: 3ul FISSEQ_ppRCA in 597 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 513
10X Phi29 Buffer 60
25mM dNTP 6
2mM aa-dUTP 12
Phi29 9
Total 600
  • Wash 1X PBS once
  • 12ul BS(PEG)9 in 588ul 1X PBS
    • Incubate @RT 1hr
  • Wash 1X PBS twice
  • Quench with 1M Tris pH 8.0 @RT 1hr
  • Wash 1X PBS twice
  • Add 1uM dcProbe1-Cy3 in 30% formamide + 2X SSC
    • Preheat to 75C for 5min
    • Incubate @RT for 10min
  • Wash 2X SSC twice
  • Image
    • Used Pinhole: 2.0

Results

  • Saved in 5-8-2014