Matt:LabNotes/2014-3-26: Difference between revisions
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>Mzcai m (→Secondary RCA) |
>Mzcai m (→Results) |
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(One intermediate revision by the same user not shown) | |||
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*Washed twice with 2X SSC | *Washed twice with 2X SSC | ||
*'''Saved in 3-28-2014''' | *'''Saved in 3-28-2014''' | ||
**Imaged with 5% laser and Gain of 550 & 650 | |||
===Results=== | |||
*Using Gain of 550 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Image''' | |||
| align="center" style="background:#f0f0f0;"|'''pp Concentration [pM]''' | |||
| align="center" style="background:#f0f0f0;"|'''Rolonies''' | |||
|- | |||
| 'Pos1_NegCtrl_Cy3_ch00_MIP.tif'||0||5963 | |||
|- | |||
| 'Pos2_NegCtrl_Cy3_ch00_MIP.tif'||0||9467 | |||
|- | |||
| 'Pos3_10pM_Cy3_ch00_MIP.tif'||10||9439 | |||
|- | |||
| 'Pos4_10pM_Cy3_ch00_MIP.tif'||10||6314 | |||
|- | |||
| 'Pos5_50pM_Cy3_ch00_MIP.tif'||50||6502 | |||
|- | |||
| 'Pos6_50pM_Cy3_ch00_MIP.tif'||50||6789 | |||
|- | |||
| 'Pos7_100pM_Cy3_ch00_MIP.tif'||100||6967 | |||
|- | |||
| 'Pos8_100pM_Cy3_ch00_MIP.tif'||100||4796 | |||
|- | |||
| 'Pos11_10nM_Cy3_ch00_MIP.tif'||10000||2167 | |||
|- | |||
| 'Pos12_10nM_Cy3_ch00_MIP.tif'||10000||6316 | |||
|} | |||
*Using Gain of 650 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Image''' | |||
| align="center" style="background:#f0f0f0;"|'''pp Concentration [pM]''' | |||
| align="center" style="background:#f0f0f0;"|'''Rolonies''' | |||
|- | |||
| 'Pos3_10pM_G650_Cy3_ch00_MIP.tif'||10||6310 | |||
|- | |||
| 'Pos4_10pM_G650_Cy3_ch00_MIP.tif'||10||6494 | |||
|- | |||
| 'Pos5_50pM_G650v2_Cy3_ch00_MIP.tif'||50||6789 | |||
|- | |||
| 'Pos6_50pM_G650_Cy3_ch00_MIP.tif'||50||6971 | |||
|- | |||
| 'Pos7_100pM_G650_Cy3_ch00_MIP.tif'||100||4796 | |||
|- | |||
| 'Pos8_100pM_G650_Cy3_ch00_MIP.tif'||100||4986 | |||
|- | |||
| 'Pos10_1nM_G650_Cy3_ch00_MIP.tif'||1000||2164 | |||
|- | |||
| 'Pos11_10nM_G650_Cy3_ch00_MIP.tif'||10000||6321 | |||
|- | |||
| 'Pos12_10nM_G650_Cy3_ch00_MIP.tif'||10000||5962 | |||
|} | |||
===Conclusion=== | |||
*None of the sample showed significant difference from negative control so can only assume template concentration was too low | |||
*Unlike [[Matt:LabNotes/2014-3-19 | last time]] where negative control had absolutely zero rolonies, the negative control showed a LOT of '''dim''' signal | |||
**The rolonies counted here had a raw intensity value of ~1,000 | |||
**The true rolonies counted last time had a raw intensity value of ~10,000 | |||
*The other samples were also '''dim''' rolonies with intensity of ~1,000 instead of the normal ~10,000 | |||
*What can cause dim signal? | |||
**Autofluorescence | |||
***But would expect to see this last time too unless difference between PGP1f and NCI-H1975 | |||
**Dye binding to fixed template | |||
***Wouldn't expect to see so many since template concentration was low | |||
***Also should've shown up in negative control last time too |
Latest revision as of 22:38, 31 March 2014
Padlock Probe Concentration Test using Low Conc (1nM) Fixed Template[edit]
- Previously fixed 10uM template resulted in more targets than realistically would be in cells
- 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 2X SSC to 85C (6ul 100nM MALAT1_Template + 60ul 20X SSC + 534ul H2O)
- 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[edit]
- 1X Ampligase Buffer
- 0.5 U/ul Ampligase
- Incubate @ 45C for 20 hours
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)
- 600ul Total Volume
- Add 100ul each well and incubate 20hrs at 30C. (Put in at 1:30pm 3/27/14)
- 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 3-28-2014
- Imaged with 5% laser and Gain of 550 & 650
Results[edit]
- Using Gain of 550
Image | pp Concentration [pM] | Rolonies |
'Pos1_NegCtrl_Cy3_ch00_MIP.tif' | 0 | 5963 |
'Pos2_NegCtrl_Cy3_ch00_MIP.tif' | 0 | 9467 |
'Pos3_10pM_Cy3_ch00_MIP.tif' | 10 | 9439 |
'Pos4_10pM_Cy3_ch00_MIP.tif' | 10 | 6314 |
'Pos5_50pM_Cy3_ch00_MIP.tif' | 50 | 6502 |
'Pos6_50pM_Cy3_ch00_MIP.tif' | 50 | 6789 |
'Pos7_100pM_Cy3_ch00_MIP.tif' | 100 | 6967 |
'Pos8_100pM_Cy3_ch00_MIP.tif' | 100 | 4796 |
'Pos11_10nM_Cy3_ch00_MIP.tif' | 10000 | 2167 |
'Pos12_10nM_Cy3_ch00_MIP.tif' | 10000 | 6316 |
- Using Gain of 650
Image | pp Concentration [pM] | Rolonies |
'Pos3_10pM_G650_Cy3_ch00_MIP.tif' | 10 | 6310 |
'Pos4_10pM_G650_Cy3_ch00_MIP.tif' | 10 | 6494 |
'Pos5_50pM_G650v2_Cy3_ch00_MIP.tif' | 50 | 6789 |
'Pos6_50pM_G650_Cy3_ch00_MIP.tif' | 50 | 6971 |
'Pos7_100pM_G650_Cy3_ch00_MIP.tif' | 100 | 4796 |
'Pos8_100pM_G650_Cy3_ch00_MIP.tif' | 100 | 4986 |
'Pos10_1nM_G650_Cy3_ch00_MIP.tif' | 1000 | 2164 |
'Pos11_10nM_G650_Cy3_ch00_MIP.tif' | 10000 | 6321 |
'Pos12_10nM_G650_Cy3_ch00_MIP.tif' | 10000 | 5962 |
Conclusion[edit]
- None of the sample showed significant difference from negative control so can only assume template concentration was too low
- Unlike last time where negative control had absolutely zero rolonies, the negative control showed a LOT of dim signal
- The rolonies counted here had a raw intensity value of ~1,000
- The true rolonies counted last time had a raw intensity value of ~10,000
- The other samples were also dim rolonies with intensity of ~1,000 instead of the normal ~10,000
- What can cause dim signal?
- Autofluorescence
- But would expect to see this last time too unless difference between PGP1f and NCI-H1975
- Dye binding to fixed template
- Wouldn't expect to see so many since template concentration was low
- Also should've shown up in negative control last time too
- Autofluorescence