Matt:LabNotes/2013-12-9: Difference between revisions

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*Background: Agi26k0gap_ShotinDark.lif_Pre-dcProbe4_background_Cy3
*Background: Agi26k0gap_ShotinDark.lif_Pre-dcProbe4_background_Cy3
**Count = 11
**Count = 175 (MAJORITY are from large bright spots that shouldn't count because non-specific)
[[File:Agi26k0gap_ShotinDark.lif_Pre-dcProbe4_background_Cy3_ch00_MIP_PISA7Overlay.png|450px]]
[[File:Agi26k0gap_ShotinDark.lif_Pre-dcProbe4_background_Cy3_ch00_MIP_PISA7Overlay.png|450px]]
*'''dcProbe4''' (Gain 680): Agi26k0gap_ShotinDark.lif_SecondaryRolony_dcProbe4_L5_Gain680
*'''dcProbe4''' (Gain 680): Agi26k0gap_ShotinDark.lif_SecondaryRolony_dcProbe4_L5_Gain680
Line 90: Line 90:


*Background: Agi26k0gap_ShotinDark.lif_Pre-dcProbe3_background_Cy3
*Background: Agi26k0gap_ShotinDark.lif_Pre-dcProbe3_background_Cy3
**Count = 175 (MAJORITY are from large bright spots that shouldn't count because non-specific)
**Count = 11
[[File:Agi26k0gap_ShotinDark.lif_Pre-dcProbe3_background_Cy3_ch00_MIP_PISA7Overlay.png|450px]]
[[File:Agi26k0gap_ShotinDark.lif_Pre-dcProbe3_background_Cy3_ch00_MIP_PISA7Overlay.png|450px]]
*'''dcProbe3''' (Laser 3%): Agi26k0gap_ShotinDark.lif_SecondaryRolony_dcProbe3_L3_Gain680
*'''dcProbe3''' (Laser 3%): Agi26k0gap_ShotinDark.lif_SecondaryRolony_dcProbe3_L3_Gain680

Revision as of 23:30, 17 December 2013

Using Agilent 0 gap Probes to Generate Secondary Rolonies

Probeset Used

  • Agi26k0gap Probes - 58.6ul of 28.4ng/ul (Matt:LabNotes/2013-6-27)
    • 28.4ng/ul = 28.4 ng/ul x 1/(193nt x 325 Da/nt) x 10^-9 g/ng x 10^6 ul/L = 453 pM
    • Dilute volume to 100ul with 41.4ul of H2O
      • Working Probe Conc.: 265 pM
        • ~0.02 pM for each probe (12,964 probes in probeset)
  • Better than using CA12k probeset because they have decoding barcodes shortened from 22nt to 20nt (and our dcProbe#-Cy3 are 22nt)

Dye-Probes Used

  • Used dcProbe#-Cy3 (Sequences found: Hosuk:FISSEQ_Primer_information#Detection_Probes
    • Because the probe sequences match (NOT complementary) to barcode region of padlock probes, I must do secondary rolony generation to detect
      • To detect padlock probe hybridization could use Shifted FISSEQ_2ndRCAprimerDye to detect all padlock probes but then would have to strip and re-hybridize (20 hr process) before doing secondary rolony generation

How many probes in Agi26k0gap probeset contain barcode region for each dye-probe

Dye-probe Number of probes detectable
dcProbe0-Cy3 3,641
dcProbe1-Cy3 2,894
dcProbe2-Cy3 2,950
dcProbe3-Cy3 2,910
dcProbe4-Cy3 3,014

Experimental Procedure

  • Using dish 11/11 #2
  • Add FISSEQ_Adpt (Cy3) in 30% formamide and 2X SSC
    • Image: Agi26k0gap_ShotinDark.lif_PrimaryRolonyCy3_Laser5_Gain680_
    • Image: Agi26k0gap_ShotinDark.lif_PrimaryRolonyCy3_Laser3_Gain600_
  • Strip with 80% formamide
  • Add 100ul of 265pM Agi26k0gap probes (preheat to 80C for 10 min) and incubate at 50C for 20 hours (6pm Friday to 2pm Saturday)
    • Even though sealed with moist towel in bag, still dried up
  • Wash with PBS once
  • Add 100ul Ampligase (5U total) and incubate at 50C for 19 hours (2:30pm Saturday to 9:30am Sunday)
  • Wash with PBS once
  • Pre-anneal RCA primer (1uM FISSEQ_ppRCA in 2X SSC/30% formamide pre-heated to 60C)
    • Incubate at 60C for 15 min
    • Wash with 2X SSC twice and then 0.1X SSC twice
  • RCA at 30C for 20.5 hours (10:15am Sunday to 6:45am Monday)
    • 171ul H2O, 20ul 10X Phi29 buffer, 2ul 25mM dNTP, 4ul 2mM aa-dUTP, 3ul Phi29 (0.1ug/ul)Polymerase
  • Wash with PBS once
  • Add 1ml BS(PEG)9 for 1 hour at RT
  • Wash with PBS twice
  • Add 200ul 1M Tris pH 8.0 for 30 min at RT
  • Rinse with PBS thrice
  • Image background: Agi26k0gap_ShotinDark.lif_Pre-dcProbe4_background_Cy3
    • Cy3 channel even though NO dye of any kind was added
  • Add dcProbe4-Cy3 in 30% formamide and 2X SSC
  • Image: Agi26k0gap_ShotinDark.lif_SecondaryRolony_dcProbe4_L5_Gain680
  • Image: Agi26k0gap_ShotinDark.lif_SecondaryRolony_dcProbe4_L5_Gain712
  • Strip with 80% formamide
  • Image: Agi26k0gap_ShotinDark.lif_Pre-dcProbe3_background_Cy3
  • Add dcProbe3-Cy3 in 30% formamide and 2X SSC
  • Image: Agi26k0gap_ShotinDark.lif_SecondaryRolony_dcProbe3_L3_Gain680
    • Laser 3%
  • Image: Agi26k0gap_ShotinDark.lif_SecondaryRolony_dcProbe3_L5_Gain680
    • Laser 5%

Image Analysis

  • Primary Rolonies: Agi26k0gap_ShotinDark.lif_PrimaryRolonyCy3_Laser3_Gain600_
    • Used this image because less saturated
    • PISA7: Gaussian: 3, Bkgmult lower: 7
      • Using pixel area 4-100 is better as seen below, it counts some of the larger circles that are obviously rolonies
Area: 4-49 Area: 4-100
Count: 3,789 Count: 3,833
File:Agi26k0gap ShotinDark.lif PrimaryRolonyCy3 Laser3 Gain600 ch00 MIP PISA7Overlay 4-49.png File:Agi26k0gap ShotinDark.lif PrimaryRolonyCy3 Laser3 Gain600 ch00 MIP PISA7Overlay 4-100.png
  • Background: Agi26k0gap_ShotinDark.lif_Pre-dcProbe4_background_Cy3
    • Count = 175 (MAJORITY are from large bright spots that shouldn't count because non-specific)

File:Agi26k0gap ShotinDark.lif Pre-dcProbe4 background Cy3 ch00 MIP PISA7Overlay.png

  • dcProbe4 (Gain 680): Agi26k0gap_ShotinDark.lif_SecondaryRolony_dcProbe4_L5_Gain680
    • Count = 17

File:Agi26k0gap ShotinDark.lif SecondaryRolony dcProbe4 L5 Gain680 ch00 MIP PISA7Overlay.png

  • dcProbe4 (Gain 712): Agi26k0gap_ShotinDark.lif_SecondaryRolony_dcProbe4_L5_Gain712
  • Background: Agi26k0gap_ShotinDark.lif_Pre-dcProbe3_background_Cy3
    • Count = 11

File:Agi26k0gap ShotinDark.lif Pre-dcProbe3 background Cy3 ch00 MIP PISA7Overlay.png

  • dcProbe3 (Laser 3%): Agi26k0gap_ShotinDark.lif_SecondaryRolony_dcProbe3_L3_Gain680
    • Count = 137 (125 "real" aka not part of a larger spot of non-specific binding)

File:Agi26k0gap ShotinDark.lif SecondaryRolony dcProbe3 L3 Gain680 ch00 MIP PISA7Overlay.png

  • dcProbe3 (Laser 5%): Agi26k0gap_ShotinDark.lif_SecondaryRolony_dcProbe3_L5_Gain680