Hosuk:LabNotes/2013-11-12: Difference between revisions
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>Hosuki78 (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 | *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==== | ====Padlock probe concentration==== | ||
*2uM , 0.66uM was working | *2uM , 0.66uM was working | ||
*1nM | *1nM --> 12x10^10 numbers | ||
*MALAT1 was roughly 3700 numbers in one field of view (1mm^2) | *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 | *--> if there are 100 targets in Rolony --> 6.5 x 10^6 targets | ||
*0.1nM, 1nM, 10nM, 100nM, 1uM , 5uM for 1 padlock probes | *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 | *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 | ||
Line 26: | Line 27: | ||
**Time : 4hr | **Time : 4hr | ||
**Enzyme concentration : 5U (1uL of Ampligase) | **Enzyme concentration : 5U (1uL of Ampligase) | ||
*Image 1st rolony --> Image padlock probes --> Image 2nd Rolony | |||
*will be done in next week | |||
*Optimize --> later | *Optimize --> later |
Latest revision as of 18:09, 18 November 2013
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