Matt:LabNotes/2015-3-31: Difference between revisions
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>Mzcai |
>Mzcai m (→Slide PCR) |
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*Place cover-slip on clean glass slide (tissue face up) | *Place cover-slip on clean glass slide (tissue face up) | ||
*Use | *Use silicon isolator with circular opening to seal tissue | ||
**Neg Ctrl | |||
** | |||
*Add 100ul PCR mix and seal with plastic to prevent evaporation | *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 | ||
*Remove plastic seal and pipette solution into PCR tubes | *Remove plastic seal and pipette solution into PCR tubes | ||
**E | |||
*Add 100ul H2O, let sit for 5min, and pipette into empty PCR tubes (to get more amplicon) | *Add 100ul H2O, let sit for 5min, and pipette into empty PCR tubes (to get more amplicon) | ||
Revision as of 21:57, 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 silicon isolator with circular opening to seal tissue
- Neg Ctrl
- 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
- E
- Add 100ul H2O, let sit for 5min, and pipette into empty PCR tubes (to get more amplicon)
Add Sequencing Adapters
PCR Test
Components | 1X Volume | 3X Volume |
Slide PCR amplicon | 5 | 0 |
SeqRolony_P5 | 0.4 | 1.2 |
SeqRolony_P7 | 0.4 | 1.2 |
2X KAPA SYBG MM | 12.5 | 37.5 |
H2O | 6.7 | 20.1 |
Total | 25 | 60 |
- Aliquot 20ul from 3X master mix and add 5ul slide amplicon
Program 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
[[ | 650px]]
PCR
Components | 1X Volume | 3X Volume |
Slide PCR amplicon | 5 | 0 |
SeqRolony_P5 | 0.4 | 1.2 |
SeqRolony_P7 | 0.4 | 1.2 |
2X KAPA SYBG MM | 12.5 | 37.5 |
H2O | 6.7 | 20.1 |
Total | 25 | 60 |