Matt:LabNotes/2014-3-31: Difference between revisions
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>Mzcai (Created page with "==Padlock Probe Concentration Test using Med Conc (100nM) Fixed Template== * Previously fixed 1nM template resulted in almost no rolonies *96-wel...") |
>Mzcai m (→Results) |
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(8 intermediate revisions by the same user not shown) | |||
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*0.5 U/ul Ampligase | *0.5 U/ul Ampligase | ||
*Incubate @ 45C for | *Incubate @ 45C for 21 hours | ||
====Negative Control 0pM ( | ====Negative Control 0pM (C7&C8)==== | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
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|} | |} | ||
====10pM ( | ====10pM (C9&C10)==== | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
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|} | |} | ||
====50pM ( | ====50pM (F3&F4)==== | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
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|} | |} | ||
====100pM ( | ====100pM (F5&F6)==== | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
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|} | |} | ||
====1nM ( | ====1nM (F7&F8)==== | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
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|} | |} | ||
====10nM ( | ====10nM (F9&F10)==== | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
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| Total||100 | | 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 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. | |||
##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 50ul each well and incubate 20hrs at 30C. | |||
#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 650ul of 1uM dcProbe2-Cy3 (643.5ul 2XSSC & 30%formmaide + 6.5ul 100uM dcProbe2-Cy3) | |||
**Extra volume since some always evaporates when heated | |||
*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 4-2-2014''' | |||
===Results=== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Position''' | |||
| align="center" style="background:#f0f0f0;"|'''Well''' | |||
| align="center" style="background:#f0f0f0;"|'''Concentration''' | |||
| align="center" style="background:#f0f0f0;"|'''Rolony Count''' | |||
|- | |||
| Pos1||C7||0pM||0 | |||
|- | |||
| Pos2||C8||0pM||0 | |||
|- | |||
| Pos3||C9||10pM||2458 | |||
|- | |||
| Pos4||C10||10pM||1952 | |||
|- | |||
| Pos5||F3||50pM||15805 | |||
|- | |||
| Pos6||F4||50pM||7253 | |||
|- | |||
| Pos7||F5||100pM||7754 | |||
|- | |||
| Pos8||F6||100pM||6210 | |||
|- | |||
| Pos9||F7||1nM||14548 | |||
|- | |||
| Pos10||F8||1nM||13323 | |||
|- | |||
| Pos11||F9||10nM||27904 | |||
|- | |||
| Pos12||F10||10nM||22660 | |||
|} | |||
====Compare these results with [[Matt:LabNotes/2014-3-19 | 10uM Template]]==== | |||
*In red: Previous concentration test where 10uM template in 2X SSC was fixed | |||
**Although a higher concentration, I think 2X SSC interferes with BS(PEG)9 fixation hence the actual lower concentration | |||
*In blue: This concentration test results | |||
[[File:PpConcTest_04042014.png]] |
Latest revision as of 01:07, 5 April 2014
Padlock Probe Concentration Test using Med Conc (100nM) Fixed Template[edit]
- Previously fixed 1nM template resulted in almost no rolonies
- 96-well plate PGP1f Cells
- Fixed 12/26/13 and RT with 12/30/13
- Using wells C7-C10, F3-F10
- Marked 1 position for each well (adapter clips on bottom&right)
- .maf file saved in 3-31-2014
- Test padlock probe concentrations NegCtrl-0pM, 10pM, 50pM, 100pM, 1nM, 10nM
Fixing Template in cells[edit]
- Pre-heat 100nM MALAT1_Template to 85C
- Add 50ul to each well
- Incubate @RT for 15min
- Aspirate but don't wash
- Add 50ul 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 21 hours
Negative Control 0pM (C7&C8)[edit]
Component | Volume |
10X Ampligase Buffer | 10 |
5U/ul Ampligase | 10 |
H2O | 80 |
Total | 100 |
10pM (C9&C10)[edit]
Component | Volume |
10X Ampligase Buffer | 10 |
1nM ppMALAT1 | 1 |
5U/ul Ampligase | 10 |
H2O | 79 |
Total | 100 |
50pM (F3&F4)[edit]
Component | Volume |
10X Ampligase Buffer | 10 |
1nM ppMALAT1 | 5 |
5U/ul Ampligase | 10 |
H2O | 75 |
Total | 100 |
100pM (F5&F6)[edit]
Component | Volume |
10X Ampligase Buffer | 10 |
1nM ppMALAT1 | 10 |
5U/ul Ampligase | 10 |
H2O | 70 |
Total | 100 |
1nM (F7&F8)[edit]
Component | Volume |
10X Ampligase Buffer | 10 |
10nM ppMALAT1 | 10 |
5U/ul Ampligase | 10 |
H2O | 70 |
Total | 100 |
10nM (F9&F10)[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 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.
- 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 50ul each well and incubate 20hrs at 30C.
- 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[edit]
- Made 650ul of 1uM dcProbe2-Cy3 (643.5ul 2XSSC & 30%formmaide + 6.5ul 100uM dcProbe2-Cy3)
- Extra volume since some always evaporates when heated
- 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 4-2-2014
Results[edit]
Position | Well | Concentration | Rolony Count |
Pos1 | C7 | 0pM | 0 |
Pos2 | C8 | 0pM | 0 |
Pos3 | C9 | 10pM | 2458 |
Pos4 | C10 | 10pM | 1952 |
Pos5 | F3 | 50pM | 15805 |
Pos6 | F4 | 50pM | 7253 |
Pos7 | F5 | 100pM | 7754 |
Pos8 | F6 | 100pM | 6210 |
Pos9 | F7 | 1nM | 14548 |
Pos10 | F8 | 1nM | 13323 |
Pos11 | F9 | 10nM | 27904 |
Pos12 | F10 | 10nM | 22660 |
Compare these results with 10uM Template[edit]
- In red: Previous concentration test where 10uM template in 2X SSC was fixed
- Although a higher concentration, I think 2X SSC interferes with BS(PEG)9 fixation hence the actual lower concentration
- In blue: This concentration test results