Matt:LabNotes/2015-8-18: Difference between revisions
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>Mzcai (Created page with "=BA8 Section DARTFISH suppv2 with Fiducial Beads= *Similar to Matt:LabNotes/2015-8-4 but with suppressor oligos and 5 cycle decoding *3-4 month old BA8 tissue section *su...") |
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=BA8 Section DARTFISH suppv2 with Fiducial Beads= | =BA8 Section DARTFISH suppv2 with Fiducial Beads= | ||
* | *Repeat [[Matt:LabNotes/2015-8-17]] but with fresh PFA and 0.01N HCl (instead of 0.1N during pepsin step) to see if that helps with tissue degradation | ||
*3-4 month old BA8 tissue section | *3-4 month old BA8 tissue section | ||
*suppv2 design | *suppv2 design | ||
*Use FluoSpheres Amine-Modified Microspheres 0.2um (505/515) as fiducial markers | *Use FluoSpheres Amine-Modified Microspheres 0.2um (505/515) as fiducial markers | ||
*Try only 5min incubation with TX-500 to see if that decreases tissue degradation | *Try only 5min incubation with TX-500 to see if that decreases tissue degradation | ||
==DARTFISH on BA8== | ==DARTFISH on BA8== | ||
#Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate | #Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate | ||
#*EtOH sterilize and UV | #*EtOH sterilize and UV | ||
# | #Made 40ml 4% PFA at RT | ||
#*10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O | |||
#Take out BA8 section from -80C and incubate on 50C hot plate for 3min | #Take out BA8 section from -80C and incubate on 50C hot plate for 3min | ||
#Submerge section in 5ml 4% PFA in 6 well culture plate for 15min at 37C | #Submerge section in 5ml 4% PFA in 6 well culture plate for 15min at 37C | ||
#Wash with 2X SSPE by submerging in 2 clean wells of culture plate with 5ml SSPE | #Wash with 2X SSPE by submerging in 2 clean wells of culture plate with 5ml SSPE | ||
#Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive | #Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive | ||
#Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT | #Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT | ||
#*New solution made by Justin | #*New solution made by Justin | ||
#Wash with nuclease-free H2O three times | #Wash with nuclease-free H2O three times | ||
#Add 0.01% Pepsin in 0.1N HCl and incubate for 5min at 37C | #Add 0.01% Pepsin in 0.1N HCl and incubate for 5min at 37C | ||
#*2ul 1%Pepsin + | #*2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O | ||
#Wash with nuclease-free PBS three times | #Wash with nuclease-free PBS three times | ||
#Prepare Reverse Transcription Mix '''on ice''' | #Prepare Reverse Transcription Mix '''on ice''' | ||
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| Total||200 | | Total||200 | ||
|} | |} | ||
#Incubate 10min at 4C and then ~18hr at 37C | #Incubate 10min at 4C and then ~18hr at 37C<!-- | ||
#Wash with 1X PBS once | #Wash with 1X PBS once | ||
#Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate 1hr at RT | #Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate 1hr at RT |
Revision as of 22:14, 18 August 2015
BA8 Section DARTFISH suppv2 with Fiducial Beads
- Repeat Matt:LabNotes/2015-8-17 but with fresh PFA and 0.01N HCl (instead of 0.1N during pepsin step) to see if that helps with tissue degradation
- 3-4 month old BA8 tissue section
- suppv2 design
- Use FluoSpheres Amine-Modified Microspheres 0.2um (505/515) as fiducial markers
- Try only 5min incubation with TX-500 to see if that decreases tissue degradation
DARTFISH on BA8
- Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate
- EtOH sterilize and UV
- Made 40ml 4% PFA at RT
- 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O
- Take out BA8 section from -80C and incubate on 50C hot plate for 3min
- Submerge section in 5ml 4% PFA in 6 well culture plate for 15min at 37C
- Wash with 2X SSPE by submerging in 2 clean wells of culture plate with 5ml SSPE
- Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
- Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT
- New solution made by Justin
- Wash with nuclease-free H2O three times
- Add 0.01% Pepsin in 0.1N HCl and incubate for 5min at 37C
- 2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O
- Wash with nuclease-free PBS three times
- Prepare Reverse Transcription Mix on ice
Components | Volume |
H2O | 157 |
10X M-MuLV Buffer | 20 |
25mM dNTP | 2 |
2mM aa-dUTP | 4 |
100uM N9 | 5 |
RNase Inhibitor | 2 |
M-MuLV RTase | 10 |
Total | 200 |
- Incubate 10min at 4C and then ~18hr at 37C