Matt:LabNotes/2013-12-9

From ZhangLabWiki
Revision as of 00:50, 10 December 2013 by >Mzcai (Created page with "===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....")
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search

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: 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: