Revision as of 22:36, 25 March 2014 by >Mzcai
Padlock Probe Concentration Test using Fixed Template
- 96-well plate NCI-H1975 Cells (C8, C9, C10, D8, D9, D10, E8, E9, E10, F9)
- Alan already marked locations with good cell density
- Test concentrations NegCtrl-0pM, 10pM, 50pM, 100pM, 10nM
Fixing Template in cells
- Pre-heat 10uM MALAT1_Template in 2X SSC to 85C (50ul 100uM MALAT1_Template + 50ul 20X SSC + 400ul H2O)
- Add 50ul to each well
- Aspirate but don't wash
- Add 50ul BS(PEG)9 mix (10ul BS(PEG)9 + 490ul 1X PBS)
- Wash with 1X PBS twice
- Add 50ul 1M Tris pH 8.0
- Wash with 1X PBS twice
Anneal Padlock Probes
- 1X Ampligase Buffer
- 0.5 U/ul Ampligase
- Incubate @ 45C for 24 hours (Started at 4pm)
- Wrapped in parafilm and put in ziploc with wet towel
Negative Control 0pM (C8 & C9)
Component
|
Volume
|
10X Ampligase Buffer |
10
|
5U/ul Ampligase |
10
|
H2O |
80
|
Total |
100
|
10pM (D8 & D9)
Component
|
Volume
|
10X Ampligase Buffer |
10
|
1nM ppMALAT1 |
1
|
5U/ul Ampligase |
10
|
H2O |
79
|
Total |
100
|
50pM (E8 & E9)
Component
|
Volume
|
10X Ampligase Buffer |
10
|
1nM ppMALAT1 |
5
|
5U/ul Ampligase |
10
|
H2O |
75
|
Total |
100
|
100pM (C10 & D10)
Component
|
Volume
|
10X Ampligase Buffer |
10
|
1nM ppMALAT1 |
10
|
5U/ul Ampligase |
10
|
H2O |
70
|
Total |
100
|
10nM (E10 & F9)
Component
|
Volume
|
10X Ampligase Buffer |
10
|
1uM ppMALAT1 |
1
|
5U/ul Ampligase |
10
|
H2O |
79
|
Total |
100
|
Secondary RCA
- Aspirate but don't wash
- Pre-annealing the FISSEQ_ppRCA primer for the rolling circle amplification reaction.
- 2.5ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 497.5ul of 2xSSC/30% formamide
- Preheat primer at 60C (Tm = 55C when not in 30% formamide)
- Add 50ul 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.
- 427.5 ul ddH2O,
- 50 ul 10x Phi29 buffer,
- 5 ul 25 mM dNTP,
- 10 ul 2 mM aminoallyl dUTP
- 7.5 ul Phi29 DNA polymerase (Epicentre, low concentration)
- Add 50ul each well and incubate 20hrs at 30C. (Put in at 4:30pm 3/20/14)
- Wash 1X PBS once
- Add 50ul BS(PEG)9 mix to each well and incubate @RT for 1hr
- Wash 1X PBS twice
- Add 50ul 1M Tris pH 8.0 @RT for 30min
- Wash 1X PBS twice
Detection
- Made 500ul of 1uM dcProbe2-Cy3 (495ul 2XSSC & 30%formmaide + 5ul 100uM dcProbe2-Cy3)
- Pre-heated @75C for 5 min and then added 50ul to each well
- Let sit @RT for 5 min
- Washed twice with 2X SSC
- Saved in 3-24-2014
Results
- All imaging was done with 5% Laser and Gain of 550
- C10_100pM had a Z-thickness of ~37 microns so tried taking both 20 and 30 z-slices
- As seen in the table it didn't make a big difference
- The disparity between C10 and D10 (both 100pM) can be partly attributed to different number of cells in field of view
Image
|
Rolony Count
|
C8_NegCtrl_G550_Cy3_ch00_MIP |
0
|
C9_NegCtrl_G550_Cy3_ch00_MIP |
0
|
D8_10pM_G550_Cy3_ch00_MIP |
51
|
D9_10pM_G550_Cy3_ch00_MIP |
101
|
E8_50pM_G550_Cy3_ch00_MIP |
355
|
E9_50pM_G550_Cy3_ch00_MIP |
764
|
C10_100pM_G550_Cy3_ch00_MIP |
3216
|
D10_100pM_G550_30slices_Cy3_ch00_MIP |
521
|
D10_100pM_G550_Cy3_ch00_MIP |
497
|
E10_10nM_G550_Cy3_ch00_MIP |
17509
|
F9_10nM_G550_Cy3_ch00_MIP |
27746
|
File:PpConcTest 03242014.JPG