Matt:LabNotes/2013-12-9
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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)
- 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
- 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
- Using dish 11/11 #2
- Add FISSEQ_Adpt (Cy3) in 30% formamide and 2X SSC
- Image: PrimaryRolonyCy3
- 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:
- Cy3 channel even though NO dye of any kind was added
- Add dcProbe4-Cy3 in 30% formamide and 2X SSC
- Image:
- Strip with 80% formamide
- Image:
- Add dcProbe3-Cy3 in 30% formamide and 2X SSC
- Image: