Matt:LabNotes/2016-1-20: Difference between revisions
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#Wash with 1X PBS twice | #Wash with 1X PBS twice | ||
#*At the maximum width, width = 8mm. The border from 7-8mm is very thin | #*At the maximum width, width = 8mm. The border from 7-8mm is very thin | ||
#RNA Removal | #RNA Removal | ||
#*Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C | #*Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C |
Revision as of 01:17, 21 January 2016
DARTFISH on BA8 Sections
- [Matt:LabNotes/2015-10-29|Last time imaged with 20X objective didn't decode enough rolonies]
- 90 sec 0.01% pepsin incubation worked well
- Picked up 9 sections from Yun today: "1/19/16 Matt & Yun Vectabonded coverslips BA8 patient 1568"
- He said many of the sections had "shattering" near the superficial layers of the thicker end (The curved part of 'P')
- The best sections are in the back of the box and the worst are in the front
- The very worst had tissue wiped off one side of coverslip and then better section stuck to the other side
Protocol
Day 1
- Prepare 2 plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Prepare fresh 40ml 4% PFA at RT
- 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O?
- Take out 2 best BA8 sections (2 furthest back) from -80C and dry on 50C hot plate for 3min
- Submerge coverslip in 5ml 4% PFA in 6 well culture plate for 15min at 37C
- Wash twice with cold 1X SSPE by submerging
- Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
- Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
- Wash with cold nf-H2O three times and check for degradation
- Slightly more stripes in gray matter
- Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
- 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
- Wash with nf-1X PBS three times and check for degradation
- Prepare Reverse Transcription Mix on ice and add
Components | Volume |
H2O | 159 |
10X M-MuLV Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
100uM N9 | 5 |
RNase Inhibitor | 2 |
M-MuLV RTase | 10 |
Total | 200 |
- Incubate 10min at 4C and then ~18hr at 37C