Matt:LabNotes/2015-3-31
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Sequence in situ Rolonies via in situ Slide PCR
- Purpose: Sequence the rolonies in situ to quantify the contributions of each probe to the decoded rolonies
- ie gene GNG4 has many rolonies but are they due to a single highly efficient probe or a number of different probes
- Primer design and general method: Matt:LabNotes/2015-3-14
Protocol
- Samples:
- Exp: BA8 CA12k_Nov2014_V4 Captured Rolonies
- NegCtrl: BA8 1st (cDNA) Rolonies (from old FISSEQ protocol)
Strip hybridized dye-probes with 80% formamide pre-heated to 80C
- The tissue is on thin cover-slip placed on a glass slide
- Take cover-slip off glass slide and put in small culture dish
- Incubate for 15min at RT and then wash twice with 1X PBS
Slide PCR
- Make PCR Reaction Mix on ice
Components | Volume | 2X Volume |
100uM SeqRolony_CL_Amplify | 2 | 4 |
2X Taq Master Mix | 100 | 200 |
H2O | 98 | 196 |
Total | 200 | 400 |
- Place cover-slip on clean glass slide (tissue face up)
- Use rubber seal with circular opening to surround tissue
- Rubber has double-sided tape and use to create seal around the tissue
- Add PCR mix and seal with plastic to prevent evaporation
- Put on thermocycler with slide adapter
Program 90C 3min -> (90C 20sec -> 58C 30sec -> 68C 45sec) x 20 -> 68C 2min -> 15C hold
- Remove plastic seal and pipette solution into PCR tubes
- Add 100ul H2O, let sit for 5min, and pipette into empty PCR tubes (to get more amplicon)