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
 
(5 intermediate revisions by the same user not shown)
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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 1:30pm 3/27/14)
#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 3-28-2014'''
*'''Saved in 4-7-2014'''
**Imaged with 5% laser and Gain of 550 & 650


===Results===
===Results===
*Using Gain of 550
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Image'''
| align="center" style="background:#f0f0f0;"|'''Position'''
| align="center" style="background:#f0f0f0;"|'''pp Concentration [pM]'''
| 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_NegCtrl_Cy3_ch00_MIP.tif'||0||5963
| Pos1||A4||0||0
|-
|-
| 'Pos2_NegCtrl_Cy3_ch00_MIP.tif'||0||9467
| Pos2||A4||0||0
|-
|-
| 'Pos3_10pM_Cy3_ch00_MIP.tif'||10||9439
| Pos3||A5||10||568
|-
|-
| 'Pos4_10pM_Cy3_ch00_MIP.tif'||10||6314
| Pos4||A5||10||448
|-
|-
| 'Pos5_50pM_Cy3_ch00_MIP.tif'||50||6502
| Pos5||D2||50||415
|-
|-
| 'Pos6_50pM_Cy3_ch00_MIP.tif'||50||6789
| Pos6||D2||50||339
|-
|-
| 'Pos7_100pM_Cy3_ch00_MIP.tif'||100||6967
| Pos7||D3||100||395
|-
|-
| 'Pos8_100pM_Cy3_ch00_MIP.tif'||100||4796
| Pos8||D3||100||365
|-
|-
| 'Pos11_10nM_Cy3_ch00_MIP.tif'||10000||2167
| Pos9||D4||1000||1065
|-
|-
| 'Pos12_10nM_Cy3_ch00_MIP.tif'||10000||6316
| Pos10||D4||1000||2027
|}
 
*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
| Pos11||D5||10000||2760
|-
|-
| 'Pos4_10pM_G650_Cy3_ch00_MIP.tif'||10||6494
| Pos12||D5||10000||3012
|-
| '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
|}
|}
[[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===
*None of the sample showed significant difference from negative control so can only assume template concentration was too low
*Only the 10uM MALAT1_Template in 2X SSC shows a linear trend at low ppMALAT1 concentrations
*Unlike [[Matt:LabNotes/2014-3-19 | last time]] where negative control had absolutely zero rolonies, the negative control showed a LOT of '''dim''' signal
**It was also the only NCI-H1975 sample
**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
*It seems as the MALAT1_Template concentration goes down, the difference between 10pM ppMALAT1 and 100pM ppMALAT1 becomes less significant
*The other samples were also '''dim''' rolonies with intensity of ~1,000 instead of the normal ~10,000
**Since one round of padlock probe production results in ~10pmole, which in a 100ul reaction volume is ~10pM
*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 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]

  1. Pre-heat 1nM MALAT1_Template in H2O to 85C
  2. Add 100ul to each well
    • Incubate @RT for 15min
  3. Aspirate but don't wash
  4. Add 100ul BS(PEG)9 mix (12ul BS(PEG)9 + 588ul 1X PBS)
    • Incubate @RT for 1hr
  5. Wash with 1X PBS twice
  6. Add 50ul 1M Tris pH 8.0
    • Incubate @ RT for 30min
  7. 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]

  1. Aspirate but don't wash
  2. Pre-annealing the FISSEQ_ppRCA primer for the rolling circle amplification reaction.
    1. 3ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 597ul of 2xSSC/30% formamide
    2. Preheat primer at 60C (Tm = 55C when not in 30% formamide)
    3. Add 100ul and incubate at 45C for 15min.
    4. Aspirate, and wash using 2x SSC twice
    5. Aspirate, and wash using 0.1x SSC twice
  3. Prepare the rolling circle amplification reaction mixtures on ice.
    1. 513 ul ddH2O,
    2. 60 ul 10x Phi29 buffer,
    3. 6 ul 25 mM dNTP,
    4. 12 ul 2 mM aminoallyl dUTP
    5. 9 ul Phi29 DNA polymerase (Epicentre, low concentration)
      • 600ul Total Volume
  4. Add 100ul each well and incubate 20hrs at 30C. (Put in at 6:00pm)
  5. Wash 1X PBS once
  6. Add 100ul BS(PEG)9 mix to each well and incubate @RT for 1hr
  7. Wash 1X PBS twice
  8. Add 100ul 1M Tris pH 8.0 @RT for 30min
  9. 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