Hosuk:LabNotes/2013-11-12

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Experimental Plan[edit]

RT with 8 different RT probes[edit]

  • for RT from a specific location
  • Will be started 11/12 evening ~ finished at 11/14 (Th)
  • Pick the good wells (8 wells or 16 wells for ACTB RT probes, 4 wells for hexamer RT) out of 32 wells


Padlock probe concentration[edit]

  • 2uM , 0.66uM was working
  • 1nM --> 12x10^10 numbers
  • MALAT1 was roughly 3700 numbers in one field of view (1mm^2) --> ~ 654,900 in one MatTek dishes
  • --> if there are 100 targets in Rolony --> 6.5 x 10^6 targets
  • 0.1nM, 1nM, 10nM, 100nM, 1uM , 5uM for 1 padlock probes --> MALAT1 padlock probe
  • Use 1 MatTek Dish --> pick one of dishes from one made @11/11 (has been used so far)
  • will be done in this week


Ampligase test[edit]

  • Just Do 2nd RCA and Ampligase, then image --> 2 MatTek Dishes --> #1 and #2@10/22 (has been used so far)
    • Try 2nd RCA, and anneal 2nd RCA detection probes
    • Use 3 padlock probes
    • Parameters
    • Temperature : 55C
    • Time : 4hr
    • Enzyme concentration : 5U (1uL of Ampligase)
  • Image 1st rolony --> Image padlock probes --> Image 2nd Rolony
  • will be done in next week
  • Optimize --> later