Matt:LabNotes/2014-3-10: Difference between revisions

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#Add 200ul BS(PEG)9 mix to each well and incubate @RT for 1hr
#Add 200ul BS(PEG)9 mix to each well and incubate @RT for 1hr
#Wash 1X PBS twice
#Wash 1X PBS twice
#Add 200ul 1M Tris pH 8.0 @RT for 1hr
#Add 200ul 1M Tris pH 8.0 @RT for 30min
#Wash 1X PBS twice
#Wash 1X PBS twice



Revision as of 19:31, 12 March 2014

Agi26k0gap Secondary Rolonies Mattek Dish

  • Since we now have a successful procedure for generating Secondary Rolonies
  • Since we have optimized PGP1f sample prep by culturing without EGF (Hosuk:LabNotes/2014-3-3)
    • In Harvard Science paper (Lee, J. et al), this culture condition resulted in more mRNA and less rRNA
    • Also microarray data on GEO shows Agi26k0gap probe set targets many high expression transcripts in PGP1f cells Matt:LabNotes/2014-3-7
  • Try to generate highly multiplexed 2nd rolonies with 12k padlock probeset
    • 30.3 pmole of Agi26k0gap prepared LabNotes/2014-3-5
      • 30.3 pmole / 200ul = 0.1515 pmole/ul = 0.1515 umole/L = 151.5uM
        • 151.5uM/12,000 probes = 0.0126 uM = 12.6 nM

Procedure

  1. Strip off dye-probes Hosuk used with 80% formamide pre-heated to 75C and incubated @45C for 15min
  2. Wash with 1X PBS twice
  3. Anneal Padlock Probes @45C for 28hrs (Put in at 11:30a)
    • 1X Ampligase Buffer
    • ~12.6nM of each padlock probe
    • 0.5 U/ul Ampligase
    • Prepared everything on ice and then pre-heated to 75C for 5 min before adding
Component Volume
10X Ampligase Buffer 20
30.3 pmole Agi26k0gap 58
5U/ul Ampligase 20
H2O 102
Total 200

Secondary RCA

  1. Aspirate but don't wash
  2. Pre-annealing the FISSEQ_ppRCA primer for the rolling circle amplification reaction.
    1. 1ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 199ul of 2xSSC/30% formamide
    2. Preheat primer at 60C (Tm = 55C when not in 30% formamide)
    3. Add 200ul and incubate at 45C for 15min.
    4. Aspirate, and wash using 2x SSC twice
    5. Aspirate, and wash using 0.1x SSC twice
  3. Prepare the rolling circle amplification reaction mixtures on ice.
    1. 171 ul ddH2O,
    2. 20 ul 10x Phi29 buffer,
    3. 2 ul 25 mM dNTP,
    4. 4 ul 2 mM aminoallyl dUTP
    5. 3 ul Phi29 DNA polymerase (Epicentre, low concentration)
      • 200ul Total Volume
  4. Add and incubate 20hrs at 30C. (Put in at 4:15pm 3/11/14)
  5. Wash 1X PBS once
  6. Add 200ul BS(PEG)9 mix to each well and incubate @RT for 1hr
  7. Wash 1X PBS twice
  8. Add 200ul 1M Tris pH 8.0 @RT for 30min
  9. Wash 1X PBS twice

Detection

  • Make 100ul of 1uM dcProbe0-Cy3, dcProbe1-Cy3, and dcProbe2-Cy3 in 30% formamide and 2X SSC

Results

  • Using PISA7 Paramters: Gaussian std (3), upper bound (-2e-4), area upper (50), area lower (4), axratio lower (.6), circ upper (1.6), circ lower (.8), perim conn (8), bkgmult lower (7)


Conclusion