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