Matt:LabNotes/2014-4-5: Difference between revisions
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>Mzcai (Created page with "==Padlock Probe Concentration Test using Low Conc (1nM) in H2O Fixed Template== * Previously fixed 100nM in H2O (as opposed to 2XSSC) and ended up...") |
>Mzcai m (→Conclusion) |
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##9 ul Phi29 DNA polymerase (Epicentre, low concentration) | ##9 ul Phi29 DNA polymerase (Epicentre, low concentration) | ||
##*600ul Total Volume | ##*600ul Total Volume | ||
#Add 100ul each well and incubate 20hrs at 30C. (Put in at | #Add 100ul each well and incubate 20hrs at 30C. (Put in at 6:00pm) | ||
#Wash 1X PBS once | #Wash 1X PBS once | ||
#Add 100ul BS(PEG)9 mix to each well and incubate @RT for 1hr | #Add 100ul BS(PEG)9 mix to each well and incubate @RT for 1hr | ||
Line 150: | Line 150: | ||
*Let sit @RT for 5 min | *Let sit @RT for 5 min | ||
*Washed twice with 2X SSC | *Washed twice with 2X SSC | ||
*'''Saved in | *'''Saved in 4-7-2014''' | ||
===Results=== | ===Results=== | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''Position''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''Well''' | ||
| align="center" style="background:#f0f0f0;"|'''Padlock Probe Conc [pM]''' | |||
| align="center" style="background:#f0f0f0;"|'''Rolonies''' | | align="center" style="background:#f0f0f0;"|'''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 [[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=== | ||
* | *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 | |||
* | |||
** |
Latest revision as of 00:30, 10 April 2014
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
- 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 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)
- 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[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)
- 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
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