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===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''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Dye-probe''' | align="center" style="background:#f0f0f0;"|'''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 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Area: 4-49''' | align="center" style="background:#f0f0f0;"|'''Area: 4-100''' |- | Count: 3,789||Count: 3,833 |- | [[File:Agi26k0gap_ShotinDark.lif_PrimaryRolonyCy3_Laser3_Gain600_ch00_MIP_PISA7Overlay_4-49.png|450px]]||[[File:Agi26k0gap_ShotinDark.lif_PrimaryRolonyCy3_Laser3_Gain600_ch00_MIP_PISA7Overlay_4-100.png|450px]] |} *Background: Agi26k0gap_ShotinDark.lif_Pre-dcProbe4_background_Cy3 **Count = 175 (MAJORITY are from large bright spots that shouldn't count because non-specific) **Location is shifted up ~half a frame compared to all other images in this experiment so not true background [[File:Agi26k0gap_ShotinDark.lif_Pre-dcProbe4_background_Cy3_ch00_MIP_PISA7Overlay.png|450px]] *'''dcProbe4''' (Gain 680): Agi26k0gap_ShotinDark.lif_SecondaryRolony_dcProbe4_L5_Gain680 **Count = 17 [[File:Agi26k0gap_ShotinDark.lif_SecondaryRolony_dcProbe4_L5_Gain680_ch00_MIP_PISA7Overlay.png|450px]] *'''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|450px]] *'''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|450px]] *'''dcProbe3''' (Laser 5%): Agi26k0gap_ShotinDark.lif_SecondaryRolony_dcProbe3_L5_Gain680 ====80% formamide stripping of dcProbe3==== *Incubating sample in 80% formamide at RT for 30 min does not seem effective **Also tried incubating at RT for 1 hour {| {{table}} | align="center" style="background:#f0f0f0;"|'''30 min''' | align="center" style="background:#f0f0f0;"|'''1 hour''' |- | [[File:Agi26k0gap_ShotinDark.lif_Pre-dcProbe2_background_Cy3_strip30min_ch00_MIP.png|450px]]||[[File:Agi26k0gap_ShotinDark.lif_Pre-dcProbe2_background_Cy3_strip1hr_ch00_MIP.png|450px]] |} *1 hour at RT not enough ====Conclusion==== *While we can see some signal, much less than expected for the 13,000 padlock probes **Test to see if 0.02pM concentration is enough ([[Hosuk:LabNotes/2013-12-3|previous experiment]] only went to 0.1pM for ppMALAT1, but at that point it saw no decreaes in rolonies counted, only decreased signal intensity using Shifted FISSEQ dye) **Include postive control (ppMALAT1) with Agi26k0gap probeset next time **Test if fixing hybridized padlock probes with BS(PEG)9 will increase efficiency *Try stripping dye-probes with 80% formamide by incubating in 50C oven **Always take a before image as background
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