Matt:LabNotes/2013-12-9
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Using Agilent 0 gap Probes to Generate Secondary Rolonies[edit]
Probeset Used[edit]
- 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)
- Working Probe Conc.: 265 pM
- Better than using CA12k probeset because they have decoding barcodes shortened from 22nt to 20nt (and our dcProbe#-Cy3 are 22nt)
Dye-Probes Used[edit]
- 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
- Because the probe sequences match (NOT complementary) to barcode region of padlock probes, I must do secondary rolony generation to detect
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[edit]
- 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[edit]
- 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)
- 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
- 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
80% formamide stripping of dcProbe3[edit]
- Incubating sample in 80% formamide at RT for 30 min does not seem effective
- Also tried incubating at RT for 1 hour
30 min | 1 hour |
File:Agi26k0gap ShotinDark.lif Pre-dcProbe2 background Cy3 strip30min ch00 MIP.png | File:Agi26k0gap ShotinDark.lif Pre-dcProbe2 background Cy3 strip1hr ch00 MIP.png |
- 1 hour at RT not enough
Conclusion[edit]
- 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 (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