Matt:LabNotes/2014-1-24

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Generating Primary Rolonies in 32 Wells[edit]

  • Hosuk fixed PGP1F cells in 32 wells (rows C-F, columns 3-10) of 96 well plate
  • I followed standard procedures to generate primary rolonies
    • Used half the concentration of Riboshredder because there wasn't enough left
  • Columns 3-8 used random hexamer for RT
  • Columns 9-10 used primers that are complementary to RAB7A mRNA a known distance from TSS
    • Row C: 220bp from TSS
    • Row D: 300bp from TSS
    • Row E: 510bp from TSS
    • Row F: 1000bp from TSS

RT RAB7A Results[edit]

  • Previously Hosuk (using FISSEQ_Adpt dye probe)found that primers of different distances from RAB7A TSS produced different numbers of primary rolonies
    • Increasing distance from TSS correlated with increasing # of primary rolonies with 1000bp having even more rolonies than produced by random hexamer primer
    • However using a dye probe specific for RAB7A target (RAB7A_1stRolonyFISH) which should be on all RAB7A primary rolonies since it is within 100bp of TSS no signal was found
      • This is why experiment is worth repeating

Checked here: Matt:LabNotes/2014-1-28