Hosuk:LabNotes/2014-9-30: Difference between revisions

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>Hosuki78
(Created page with "*LabNote ===ppCOL1A1 + Suppressor test=== *Sample : 4 dishes PGP1F, 1st Rolonies made at 2014-07-17 *Check 1st rolonies of all 5 dishes *S1, S4 --> ppCO...")
 
>Hosuki78
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*S2, S5 --> ppCOL1A1 only
*S2, S5 --> ppCOL1A1 only
*Add suppressor 10 times more than ppCOL1A1
*Add suppressor 10 times more than ppCOL1A1
[[File:Fig_MIP_PGP1F_RCA_2014-07-17_1stRolony.png.png|900px]]





Revision as of 23:09, 30 September 2014


ppCOL1A1 + Suppressor test

  • Sample : 4 dishes PGP1F, 1st Rolonies made at 2014-07-17
  • Check 1st rolonies of all 5 dishes
  • S1, S4 --> ppCOL1A1 + suppCOL1A1
  • S2, S5 --> ppCOL1A1 only
  • Add suppressor 10 times more than ppCOL1A1


File:Fig MIP PGP1F RCA 2014-07-17 1stRolony.png.png


Procedure

  • ppCOL1A1 + supp_COL1A1 : S1, S4
Components Volume
5 U/ul Ampligase 10
10X Ampligase Buffer 10
100uM ppCOL1A1 2
100uM Supp_COL1A1 20
H2O 58
Total 100


  • ppCOL1A1 : S2, S5
Components Volume
5 U/ul Ampligase 10
10X Ampligase Buffer 10
100uM ppCOL1A1 2
100uM Supp_COL1A1 0
H2O 78
Total 100


  • Incubate at 37C for 30min
  • Incubate at 60C for 24hrs
  • 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 (1000 U/µl) : 0.5uL
  • Wash with 1X PBS once
  • Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) at RT for 1hr
  • Wash with 1X PBS twice
  • Add 200ul 1M Tris pH 8.0 at RT for 30min
  • Wash with 1X PBS twice and store in PBS