Matt:LabNotes/2014-3-10
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Agi26k0gap Secondary Rolonies Mattek Dish
- Since we now have a successful procedure for generating Secondary Rolonies
- Since we have optimized PGP1f sample prep by culturing without EGF (Hosuk:LabNotes/2014-3-3)
- In Harvard Science paper (Lee, J. et al), this culture condition resulted in more mRNA and less rRNA
- Also microarray data on GEO shows Agi26k0gap probe set targets many high expression transcripts in PGP1f cells Matt:LabNotes/2014-3-7
- Try to generate highly multiplexed 2nd rolonies with 12k padlock probeset
- 30.3 pmole of Agi26k0gap prepared LabNotes/2014-3-5
- 30.3 pmole / 200ul = 0.1515 pmole/ul = 0.1515 umole/L = 151.5uM
- 151.5uM/12,000 probes = 0.0126 uM = 12.6 nM
- 30.3 pmole / 200ul = 0.1515 pmole/ul = 0.1515 umole/L = 151.5uM
- 30.3 pmole of Agi26k0gap prepared LabNotes/2014-3-5
Procedure
- Using Mattek dish #2 from Hosuk:LabNotes/2014-3-3 that was fixed in 10% formalin
- Strip off dye-probes Hosuk used with 80% formamide pre-heated to 75C and incubated @45C for 15min
- Wash with 1X PBS twice
- Anneal Padlock Probes @45C for 28hrs (Put in at 11:30a)
- 1X Ampligase Buffer
- ~12.6nM of each padlock probe
- 0.5 U/ul Ampligase
- Prepared everything on ice and then pre-heated to 75C for 5 min before adding
Component | Volume |
10X Ampligase Buffer | 20 |
30.3 pmole Agi26k0gap | 58 |
5U/ul Ampligase | 20 |
H2O | 102 |
Total | 200 |
Secondary RCA
- Aspirate but don't wash
- Pre-annealing the FISSEQ_ppRCA primer for the rolling circle amplification reaction.
- 1ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 199ul of 2xSSC/30% formamide
- Preheat primer at 60C (Tm = 55C when not in 30% formamide)
- Add 200ul and incubate at 45C for 15min.
- Aspirate, and wash using 2x SSC twice
- Aspirate, and wash using 0.1x SSC twice
- Prepare the rolling circle amplification reaction mixtures on ice.
- 171 ul ddH2O,
- 20 ul 10x Phi29 buffer,
- 2 ul 25 mM dNTP,
- 4 ul 2 mM aminoallyl dUTP
- 3 ul Phi29 DNA polymerase (Epicentre, low concentration)
- 200ul Total Volume
- Add and incubate 20hrs at 30C. (Put in at 4:15pm 3/11/14)
- Wash 1X PBS once
- Add 200ul BS(PEG)9 mix to each well and incubate @RT for 1hr
- Wash 1X PBS twice
- Add 200ul 1M Tris pH 8.0 @RT for 30min
- Wash 1X PBS twice
Detection
- Make 100ul of 1uM dcProbe0-Cy3, dcProbe1-Cy3, and dcProbe2-Cy3 in 30% formamide and 2X SSC
Results
- Using PISA7 Paramters: Gaussian std (3), upper bound (-2e-4), area upper (50), area lower (4), axratio lower (.6), circ upper (1.6), circ lower (.8), perim conn (8), bkgmult lower (7)