Hosuk:LabNotes/2013-11-12: Difference between revisions

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(Created page with "*LabNote ===Experimental Plan=== ====RT with 8 different RT probes==== *for RT from a specific location *Will be started 11/12 evening ~ finished at 11/...")
 
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*for RT from a specific location
*for RT from a specific location
*Will be started 11/12 evening ~ finished at 11/14 (Th)
*Will be started 11/12 evening ~ finished at 11/14 (Th)
*Pick the best wells out of 32 wells
*Pick the good wells (8 wells or 16 wells for ACTB RT probes, 4 wells for hexamer RT) out of 32 wells




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*2uM , 0.66uM was working
*2uM , 0.66uM was working
*1nM  12x10^10 numbers
*1nM  12x10^10 numbers
*MALAT1 was roughly 3700 numbers in one field of view (1mm^2) ~ 654,900 in one MatTek dishes
*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
*--> 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 pp
*0.1nM, 1nM, 10nM, 100nM, 1uM , 5uM for 1 padlock probes --> MALAT1 padlock probe
*1 MatTek Dish
*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====
====Ampligase test====
*Just Do 2nd RCA 2 MatTek Dishes
*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
**Try 2nd RCA, and anneal 2nd RCA detection probes
**Use 3 padlock probes
**Use 3 padlock probes
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**Time : 4hr
**Time : 4hr
**Enzyme concentration : 5U (1uL of Ampligase)
**Enzyme concentration : 5U (1uL of Ampligase)
 
*Image 1st rolony
*will be done in next week


*Optimize --> later
*Optimize --> later

Revision as of 00:48, 13 November 2013


Experimental Plan

RT with 8 different RT probes

  • 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

  • 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

  • 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
  • will be done in next week
  • Optimize --> later