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====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%
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