Matt:LabNotes/2015-3-31: Difference between revisions
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>Mzcai |
>Mzcai m (→Slide PCR) |
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| align="center" style="background:#f0f0f0;"|'''2X Volume''' | | align="center" style="background:#f0f0f0;"|'''2X Volume''' | ||
|- | |- | ||
| 100uM SeqRolony_CL_Amplify|| | | 100uM SeqRolony_CL_Amplify||1||2 | ||
|- | |- | ||
| 2X Taq Master Mix|| | | 2X Taq Master Mix||50||100 | ||
|- | |- | ||
| H2O|| | | H2O||49||98 | ||
|- | |- | ||
| Total|| | | Total||100||200 | ||
|} | |} | ||
*Place cover-slip on clean glass slide (tissue face up) | *Place cover-slip on clean glass slide (tissue face up) | ||
*Use rubber seal with circular opening to surround tissue | *Use rubber seal with circular opening to surround tissue | ||
**Rubber has double-sided tape and use to create seal around the tissue | **Rubber has double-sided tape and use to create seal around the tissue | ||
*Add PCR mix and seal with plastic to prevent evaporation | ***Had to use gap seal 200 glue to plug leaks | ||
*Add 100ul PCR mix and seal with plastic to prevent evaporation | |||
*Put on thermocycler with slide adapter | *Put on thermocycler with slide adapter | ||
'''Program''' 90C 3min -> (90C 20sec -> 58C 30sec -> 68C 45sec) x 20 -> 68C 2min -> 15C hold | '''Program''' 90C 3min -> (90C 20sec -> 58C 30sec -> 68C 45sec) x 20 -> 68C 2min -> 15C hold |
Revision as of 20:44, 31 March 2015
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 | 1 | 2 |
2X Taq Master Mix | 50 | 100 |
H2O | 49 | 98 |
Total | 100 | 200 |
- 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
- Had to use gap seal 200 glue to plug leaks
- Rubber has double-sided tape and use to create seal around the tissue
- Add 100ul 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)