Revision as of 00:30, 10 April 2014 by >Mzcai
Padlock Probe Concentration Test using Low Conc (1nM) in H2O Fixed Template[edit]
- Previously fixed 100nM in H2O (as opposed to 2XSSC) and ended up with too many templates as determined by number of rolonies generated
- Sample was previously used to fix 1nM in 2X SSC which was too low
- Assuming no template was fixed and 2 extra rounds of BS(PEG)9 has no effect we can re-use this sample
- 24-well plate PGP1f Cells cultured w/o EGF
- Fixed 3/21/14 and RT 3/21/14
- Using wells A4, A5, D2, D3, D4, D5
- Marked 2 positions for each well (adapter clips on bottom&right)
- .maf file saved in 3-26-2014
- Test padlock probe concentrations NegCtrl-0pM, 10pM, 50pM, 100pM, 1nM, 10nM
Fixing Template in cells[edit]
- Pre-heat 1nM MALAT1_Template in H2O to 85C
- Add 100ul to each well
- Aspirate but don't wash
- Add 100ul BS(PEG)9 mix (12ul BS(PEG)9 + 588ul 1X PBS)
- Wash with 1X PBS twice
- Add 50ul 1M Tris pH 8.0
- Wash with 1X PBS twice
Anneal Padlock Probes[edit]
- 1X Ampligase Buffer
- 0.5 U/ul Ampligase
- Incubate @ 45C for 24 hours (Put in at 5:30pm)
Negative Control 0pM (A4)[edit]
Component
|
Volume
|
10X Ampligase Buffer |
10
|
5U/ul Ampligase |
10
|
H2O |
80
|
Total |
100
|
10pM (A5)[edit]
Component
|
Volume
|
10X Ampligase Buffer |
10
|
1nM ppMALAT1 |
1
|
5U/ul Ampligase |
10
|
H2O |
79
|
Total |
100
|
50pM (D2)[edit]
Component
|
Volume
|
10X Ampligase Buffer |
10
|
1nM ppMALAT1 |
5
|
5U/ul Ampligase |
10
|
H2O |
75
|
Total |
100
|
100pM (D3)[edit]
Component
|
Volume
|
10X Ampligase Buffer |
10
|
1nM ppMALAT1 |
10
|
5U/ul Ampligase |
10
|
H2O |
70
|
Total |
100
|
1nM (D4)[edit]
Component
|
Volume
|
10X Ampligase Buffer |
10
|
10nM ppMALAT1 |
10
|
5U/ul Ampligase |
10
|
H2O |
70
|
Total |
100
|
10nM (D5)[edit]
Component
|
Volume
|
10X Ampligase Buffer |
10
|
1uM ppMALAT1 |
1
|
5U/ul Ampligase |
10
|
H2O |
79
|
Total |
100
|
Secondary RCA[edit]
- Aspirate but don't wash
- Pre-annealing the FISSEQ_ppRCA primer for the rolling circle amplification reaction.
- 3ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 597ul of 2xSSC/30% formamide
- Preheat primer at 60C (Tm = 55C when not in 30% formamide)
- Add 100ul 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.
- 513 ul ddH2O,
- 60 ul 10x Phi29 buffer,
- 6 ul 25 mM dNTP,
- 12 ul 2 mM aminoallyl dUTP
- 9 ul Phi29 DNA polymerase (Epicentre, low concentration)
- Add 100ul each well and incubate 20hrs at 30C. (Put in at 6:00pm)
- Wash 1X PBS once
- Add 100ul BS(PEG)9 mix to each well and incubate @RT for 1hr
- Wash 1X PBS twice
- Add 100ul 1M Tris pH 8.0 @RT for 30min
- Wash 1X PBS twice
Detection[edit]
- Made 600ul of 1uM dcProbe2-Cy3 (594ul 2XSSC & 30%formmaide + 6ul 100uM dcProbe2-Cy3)
- Pre-heated @75C for 5 min and then added 100ul to each well
- Let sit @RT for 5 min
- Washed twice with 2X SSC
- Saved in 4-7-2014
Results[edit]
Position
|
Well
|
Padlock Probe Conc [pM]
|
Rolonies
|
Pos1 |
A4 |
0 |
0
|
Pos2 |
A4 |
0 |
0
|
Pos3 |
A5 |
10 |
568
|
Pos4 |
A5 |
10 |
448
|
Pos5 |
D2 |
50 |
415
|
Pos6 |
D2 |
50 |
339
|
Pos7 |
D3 |
100 |
395
|
Pos8 |
D3 |
100 |
365
|
Pos9 |
D4 |
1000 |
1065
|
Pos10 |
D4 |
1000 |
2027
|
Pos11 |
D5 |
10000 |
2760
|
Pos12 |
D5 |
10000 |
3012
|
File:20140408 PadlockProbeConcentrationTest.JPG
- Blue is 1nM MALAT1_Template in H2O (this experiment)
- Red is 100nM MALAT1_Template in H2O (from 3-31-2014)
- Green is 10uM MALAT1_Template in 2X SSC (from 3-19-2014)
Conclusion[edit]
- Only the 10uM MALAT1_Template in 2X SSC shows a linear trend at low ppMALAT1 concentrations
- It was also the only NCI-H1975 sample
- It seems as the MALAT1_Template concentration goes down, the difference between 10pM ppMALAT1 and 100pM ppMALAT1 becomes less significant
- Since one round of padlock probe production results in ~10pmole, which in a 100ul reaction volume is ~10pM