Hosuk:LabNotes/2013-9-26

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ACTB Gene detection in 1st Rolonies[edit]

Procedure[edit]

  • Directly anneal ATTO488_ACTB_on1stRolony_target_Probe.
  • Use 06232013 sample – 3uL Phi29 (2nd rolony experiment sample) and 10uL Phi29
  • Procedure
    1. Strip Sample dishes with 80% Formamide
    2. Prepare 1uM Alex288_ACTB_on1stRolony_target_Probe in 200uL 2x SSC
    3. Preheat the probe at 65C for 5min
    4. Add probes in dish, incubate for 10min at RT
    5. Wash, and image


  • ACTB_1stRolonyFISH : GGCAAAGGCGAGGCT/3ATTO488N/
  • Shifted FISSEQ_2ndRCAprimerDye : GGATATCGGGAAGCTGAA/3ATTO488N/


Result[edit]

  • Too few, ACTB itself might not be many on entire RNAs.
  • Matt has found a data (microarray), and 5% out of RNA in PGP1F is mRNA, and 1% of mRNA is ACTB. According to this, there are very few of ACTB in 1st rolonies.


Next Plan[edit]

  • Find other gene which would be plenty in PGP1F
  • Or try RAB7A


Pictures[edit]
1st Rolonies, 06/23, 11th Try, 3uL Phi29 1st Rolonies, 06/23, 11th Try, 10uL Phi29
File:S3 3uLPhi29Enzymat 20x-NA0.8 pos01 Cy3 exp0.1-EM00.jpg File:S4 10uLPhi29Enzymat 20x-NA0.8 pos03 Cy3 exp0.1-EM5.jpg
ACTB on 1st Rolonies, 3uL Phi29 sample (raw) ACTB on 1st Rolonies, 10uL Phi29 sample (raw)
File:ACTB 1stRolonyTarget FITC 0623-3uLPhi29 pos01 fig01 FITC.jpg File:ACTB 1stRolonyTarget FITC 0623-10uLPhi29 pos02 fig01 FITC.jpg