Matt:LabNotes/2014-3-4

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Revision as of 20:42, 5 March 2014 by >Mzcai (→‎Procedure)
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3 Genes Secondary Rolonies Mattek Dish

  • Hosuk got much more primary rolonies (incl. ACTB and RAB7A) by culturing PGP1F without EGF (Hosuk:LabNotes/2014-3-3)
  • Since Matt:LabNotes/2014-2-25#Detection resulted in many MALAT1 secondary rolonies but few ACTB or RAB7A
    • This is possibly due to sample having very few ACTB or RAB7A
    • Also possible the dyes we got from Illumina (dcProbe0-Cy3 and dcProbe1-Cy3) have somehow degraded since MALAT1 detection was using a new dye-probe from IDT

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 24hrs (Put in at noon, some bubbles and hard to keep on middle glass)
    • 1X Ampligase Buffer
    • 2uM of each padlock probe
    • 0.5 U/ul Ampligase
Component Volume
10X Ampligase Buffer 20
200uM ppMALAT1 2
200uM ppACTB 2
200uM ppRAB7A 2
5U/ul Ampligase 20
H2O 154
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 1pm 3/5/14)
  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 1hr
  9. Wash 1X PBS twice