Matt:LabNotes/2015-10-7: Difference between revisions
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>Mzcai m (→Protocol) |
>Mzcai |
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=FISSEQ on Mouse Embryo Test= | =FISSEQ on Mouse Embryo Test= | ||
*Try 2 samples: 20um and 26um thickness | *Try 2 samples: A5 (20um thickness) and A6 (26um thickness) | ||
==Protocol== | ==Protocol== |
Revision as of 01:23, 8 October 2015
FISSEQ on Mouse Embryo Test
- Try 2 samples: A5 (20um thickness) and A6 (26um thickness)
Protocol
Day 1
- Sterilize culture dishes and tweezers with EtOH, RNaseZap, and then rinse with nf-H2O
- Attach 22mm x 22mm glass coverslips (with mounted sections) to bottom of culture dishes with 16mm diameter hole
- Use Double-sided tape: Adhesives Research IS-7876-35
- Wash tissue section twice using cold nf-H2O for 5min each
- Add 0.25% TX-100 in nf-H2O for 5min at RT
- Wash with cold nf-H2O twice
- Add 200ul 0.01% pepsin in 0.01N HCl (2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O) for 10min at RT
- Wash with 2ml of cold nf-PBS three times
- Prepare 2X Reverse Transcription Mix on ice
Components | Volume |
H2O | 157 |
10X M-MuLV Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
100uM FISSEQ_RT | 5 |
RNase Inhibitor | 2 |
M-MuLV RTase | 10 |
Total | 200 |
- Incubate 10min at 4C and then ~18hr at 37C
- Parafilm each dish and then put in plastic bag with wet tissue