Matt:LabNotes/2015-3-31: Difference between revisions
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>Mzcai m (→Slide PCR) |
>Mzcai |
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Line 40: | Line 40: | ||
====Add Sequencing Adapters==== | ====Add Sequencing Adapters==== | ||
====PCR Test==== | ====PCR Test==== | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|'''1X Volume''' | | align="center" style="background:#f0f0f0;"|'''1X Volume''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''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|| | | 2X KAPA SYBG MM||12.5||37.5 | ||
|- | |- | ||
| H2O|| | | H2O||6.7||20.1 | ||
|- | |- | ||
| Total||25|| | | Total||25||60 | ||
|} | |} | ||
*Aliquot | *Aliquot 20ul from 3X master mix and add 5ul slide amplicon | ||
Program | Program | ||
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min | 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min | ||
[[ | [[ | 650px]] | ||
====PCR==== | ====PCR==== | ||
Line 71: | Line 70: | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|'''1X Volume''' | | align="center" style="background:#f0f0f0;"|'''1X Volume''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''3X Volume''' | ||
|- | |- | ||
| | | Slide PCR amplicon||5||0 | ||
|- | |- | ||
| | | SeqRolony_P5||0.4||1.2 | ||
|- | |- | ||
| | | SeqRolony_P7||0.4||1.2 | ||
|- | |- | ||
| 2X KAPA SYBG MM|| | | 2X KAPA SYBG MM||12.5||37.5 | ||
|- | |- | ||
| H2O|| | | H2O||6.7||20.1 | ||
|- | |- | ||
| Total|| | | Total||25||60 | ||
|} | |}<!-- | ||
*Aliquot 86ul from | *Aliquot 86ul from 3X master mix and add 12ul captured template and 2ul corresponding reverse primer | ||
Program | Program | ||
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x13 -> 72C 3min | 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x13 -> 72C 3min |
Revision as of 20:51, 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)
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 |