Matt:LabNotes/2014-9-6: Difference between revisions

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(Created page with "=="Tertiary" Rolony Synthesis== *Hypothesis: We can increase the number of gene-specific rolonies (previously known as secondary rolonies) by repeating padlock probe capture a...")
 
>Mzcai
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*Incubate at 37C for 30min
*Incubate at 37C for 30min
*Incubate at 45C for 24hrs
*Incubate at 45C for 26hrs
*Wash with 1X PBS twice
*Add 200ul 0.5 uM FISSEQ_ppRCA primer in 2X SSC (pre-heated to 75C) and incubate at 45C for 30min
*Wash with 2X SSC twice
*Add 200ul RCA Mix and incubate at 30C for overnight
**H2O : 172uL
**10x Buffer : 20uL
**dNTP : 2uL
**a-dUTP : 4uL
**Phi29 (100 U/µl) : 2uL

Revision as of 01:17, 8 September 2014

"Tertiary" Rolony Synthesis

  • Hypothesis: We can increase the number of gene-specific rolonies (previously known as secondary rolonies) by repeating padlock probe capture and rolonization on samples with primary and secondary rolonies
    • There should be more targets for the padlock probes to capture since all primary rolonies are still fixed and 100% of secondary rolonies will be possible targets for padlock probes
    • May increase amplification bias

Procedure

  • Add 100ul Padlock Probe Mix
Components Volume
5 U/ul Ampligase 10
10X Ampligase Buffer 10
908 nM Agi26k0gap 18.6
H2O 61.4
Total 100
  • Incubate at 37C for 30min
  • Incubate at 45C for 26hrs
  • Wash with 1X PBS twice
  • Add 200ul 0.5 uM FISSEQ_ppRCA primer in 2X SSC (pre-heated to 75C) and incubate at 45C for 30min
  • Wash with 2X SSC twice
  • Add 200ul RCA Mix and incubate at 30C for overnight
    • H2O : 172uL
    • 10x Buffer : 20uL
    • dNTP : 2uL
    • a-dUTP : 4uL
    • Phi29 (100 U/µl) : 2uL