Revision as of 01:35, 1 April 2015 by >Mzcai
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
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
File:IMG 0454.JPG
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
- NegCtrl silicon had large gaps so used glue to glue all around the circle on bottom of silicon
- Exp had very small leak so used glue to seal the edges of silicon
- 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
- NegCtrl had ~90ul (good) but Exp was completely dry (bad, probably leaked out as evidenced by white haze on glass slide outside of silicon)
- Add 100ul H2O, let sit for 5min, and pipette into empty PCR tubes (to get more amplicon)
- Final amount: 190ul NegCtrl and 100ul Exp
Add Sequencing Adapters
- Qiagen PCR purification
- Nanodrop ssDNA:
- Exp: 0.6 ng/ul
- NegCtrl: 2.2 ng/ul
PCR Test
Components
|
1X Volume
|
3X Volume
|
Slide PCR amplicon |
11.7 |
0
|
SeqRolony_P5 |
0.4 |
1.2
|
SeqRolony_P7 |
0.4 |
1.2
|
2X KAPA SYBG MM |
12.5 |
37.5
|
Total |
25 |
60
|
- Aliquot 13.3ul from 3X master mix and add 11.7ul slide amplicon
Program
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
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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
|