Matt:LabNotes/2014-9-8: Difference between revisions

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(Created page with "==in situ Padlock Probe Capture Temp: 45C vs 60C== ===Procedure=== *PGP1F Samples with 1st Rolonies prepared by Hosuk **Fixed 9/6/14 and 1st Rolonies on 9/8/14 *Add 100ul Pa...")
 
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*Incubate at 37C for 30min
*Incubate at 37C for 30min
*Incubate at '''45C or 60C''' for 24hrs
*Incubate at '''45C or 60C''' for 26hrs
*Wash with 1X PBS twice
*Add 200ul 0.5uM FISSEQ_ppRCA in 2X SSC + 30% formamide (pre-heated to 75C) and incubate at 45C for 30min
*Wash with 2X SSC twice
*Add 200ul RCA Mix and incubate at 30C for 16hrs
**H2O : 172uL
**10x Buffer : 20uL
**dNTP : 2uL
**a-dUTP : 4uL
**Phi29 (100 U/µl) : 2uL

Revision as of 03:45, 10 September 2014

in situ Padlock Probe Capture Temp: 45C vs 60C

Procedure

  • PGP1F Samples with 1st Rolonies prepared by Hosuk
    • Fixed 9/6/14 and 1st Rolonies on 9/8/14
  • Add 100ul Padlock Probe Mix
Components Volume
5 U/ul Ampligase 10
10X Ampligase Buffer 10
100uM ppCOL1A1 2
H2O 78
Total 100
  • Incubate at 37C for 30min
  • Incubate at 45C or 60C for 26hrs
  • Wash with 1X PBS twice
  • Add 200ul 0.5uM FISSEQ_ppRCA in 2X SSC + 30% formamide (pre-heated to 75C) and incubate at 45C for 30min
  • Wash with 2X SSC twice
  • Add 200ul RCA Mix and incubate at 30C for 16hrs
    • H2O : 172uL
    • 10x Buffer : 20uL
    • dNTP : 2uL
    • a-dUTP : 4uL
    • Phi29 (100 U/µl) : 2uL