Matt:LabNotes/2014-4-5: Difference between revisions
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>Mzcai m (→Results) |
>Mzcai m (→Results) |
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[[File:20140408_PadlockProbeConcentrationTest.JPG]] | |||
*Blue is 1nM MALAT1_Template in H2O (this experiment) | |||
**PGP1f cells | |||
*Red is 100nM MALAT1_Template in H2O [[Matt:LabNotes/2014-3-31 | (from 3-31-2014)]] | |||
**PGP1f cells | |||
*Green is 10uM MALAT1_Template in 2X SSC [[Matt:LabNotes/2014-3-19 | (from 3-19-2014)]] | |||
**NCI-H1975 cells | |||
===Conclusion=== | ===Conclusion=== |
Revision as of 22:06, 9 April 2014
Padlock Probe Concentration Test using Low Conc (1nM) in H2O Fixed Template
- 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
- Pre-heat 1nM MALAT1_Template in H2O to 85C
- Add 100ul to each well
- Incubate @RT for 15min
- Aspirate but don't wash
- Add 100ul BS(PEG)9 mix (12ul BS(PEG)9 + 588ul 1X PBS)
- Incubate @RT for 1hr
- Wash with 1X PBS twice
- Add 50ul 1M Tris pH 8.0
- Incubate @ RT for 30min
- Wash with 1X PBS twice
Anneal Padlock Probes
- 1X Ampligase Buffer
- 0.5 U/ul Ampligase
- Incubate @ 45C for 24 hours (Put in at 5:30pm)
Negative Control 0pM (A4)
Component | Volume |
10X Ampligase Buffer | 10 |
5U/ul Ampligase | 10 |
H2O | 80 |
Total | 100 |
10pM (A5)
Component | Volume |
10X Ampligase Buffer | 10 |
1nM ppMALAT1 | 1 |
5U/ul Ampligase | 10 |
H2O | 79 |
Total | 100 |
50pM (D2)
Component | Volume |
10X Ampligase Buffer | 10 |
1nM ppMALAT1 | 5 |
5U/ul Ampligase | 10 |
H2O | 75 |
Total | 100 |
100pM (D3)
Component | Volume |
10X Ampligase Buffer | 10 |
1nM ppMALAT1 | 10 |
5U/ul Ampligase | 10 |
H2O | 70 |
Total | 100 |
1nM (D4)
Component | Volume |
10X Ampligase Buffer | 10 |
10nM ppMALAT1 | 10 |
5U/ul Ampligase | 10 |
H2O | 70 |
Total | 100 |
10nM (D5)
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.
- 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)
- 600ul Total Volume
- 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
- 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
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)
- PGP1f cells
- Red is 100nM MALAT1_Template in H2O (from 3-31-2014)
- PGP1f cells
- Green is 10uM MALAT1_Template in 2X SSC (from 3-19-2014)
- NCI-H1975 cells