Hosuk:LabNotes/2013-11-18
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Experimental Plan[edit]
Rolony in MatTek dish, 96 well plate[edit]
- MatTek dish
- Fix :
- Aspirate media, Add 10% Formalin, Incubate 37C for 15min.
- Wash with PBS 3 time, Add 2mL Ethanol
- 96 well plate
- In T25 flask, Add 1mL Trypsin, Incubate 37C for 5min.
- Add 4mL media (Total 5mL), Take 50uL, add in each well
- Fix by aspirating media, adding 10% Formalin
- Check cells after Fixation
Dye labeled Probe Annealing Test[edit]
- Check Non-specific binding, and Upper limit
- Use dcProbe#-Cy3 --> check non-specific binding
- Use Cy5-1st Rolony --> Upper limit
- Dye-Probe mixed with different concentration of Formamide
- 10%, 30%, 50%, 70% in 2x SSC
- Incubating temperature
- ??
- Check the number of Rolonies
Padlock Probe Concentration Test[edit]
- Lower limit, Upper limit of the concentration
- Normally 0.2uM for each Padlock Probe after PCR
- Use One MatTek Dish
- Use ppMALAT1
- 0.1uM, 5uM, and check the signal, then test middle concentration
- Preheat ppMALAT1 at 80C
- Incubate at 50C for 15min
Ampligase test[edit]
- Use one MatTek dish
- Add Padlock probes, Ligase, 2nd RCA and check how many 2nd Rolonies
- Ampligase reaction condition
- Add 3 Padlock probes – ppACTB, ppRAB7A, ppMALAT1
- Padlock probe concentration : 1uM each (? could be changed)
- Padlock probe incubation time : 30min
- Ampligase concentration : 5U
- Temperature : 60C
- Incubation time : 4hr.