Matt:LabNotes/2017-5-19: Difference between revisions

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=Third Try: in tube SplintR Test with formamide and ET SSB=
=Fourth Try: in tube SplintR Test with formamide=
*[[Matt:LabNotes/2017-4-22|First try showed SplintR with 10% formamide had the best sensitivity and specificity of the conditions tried]]
*[[Matt:LabNotes/2017-4-22|First try showed SplintR with 10% formamide had the best sensitivity and specificity of the conditions tried]]
*[[Matt:LabNotes/2017-5-8|Second try had unexpected trend with increasing formamide led to increased sensitivity]]
*[[Matt:LabNotes/2017-5-8|Second try had unexpected trend with increasing formamide led to increased sensitivity]]
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| align="right" | 3
| align="right" | 3
| align="right" | 0
| align="right" | 0
  | 22.5 or 21.6 (these were added before realizing wrong volume. Kept as is)
  | 22.5 or 21.6
| align="right" | 30
| align="right" | 30


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   Program
   Program
   98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min
   98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min
<!--
===Results===
==Results==
[[Media:20170520_qPCR_PPcapture_SplintRformamide.xlsx|raw data here]]<br>
[[Media:20170510_qPCR_PPcapture_SplintRformamideETSSB.xlsx|raw data here]]<br>
[[File:20170520_qPCR_PPcapture_SplintRformamide_ScatterPlot.PNG|450px]]
[[File:20170510_qPCR_PPcapture_SplintRformamideETSSB.PNG|650px]]
*Only Ampligase stands out from the rest
 
[[File:20170510_qPCR_PPcapture_SplintRformamideETSSB_ScatterPlot.PNG|450px]]
*12.5% formamide has the best sensitivity and seperation from NTC (specificity)
*BUT 0%-10% formamide should all have higher sensitivity...
 
*These results don't make sense
**First try repeating PCR with triplicates and lower conc. (maybe Zymo column purify first)
**Then try repeating experiment with concentrations that match actual conc. (100-200nM total)
 
==Repeat qPCR==
#qPCR all 16 samples 1ul each with triplicates
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''1X Volume'''
| align="center" style="background:#f0f0f0;"|'''48X Volume'''
|-
| Captured template||1||0
|-
| 10uM ISB_CA_AF||0.4||19.2
|-
| 10uM ISB_CA_AR.T2||0.4||19.2
|-
| 2X KAPA SYBG MM||12.5||600
|-
| H2O||10.7||513.6
|-
| Total||25||1,152
|}
*Aliquot 24ul from 48X master mix and add 1ul captured template


  Program
==Conclusion==
  98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x30 -> 72C 3min
*High temp denaturing beforehand definitely changed the results, theoretically removing the formamide's effect of denaturing secondary structures
*Still similar trend that isn't 100% clear...
*Need to repeat

Latest revision as of 03:23, 9 June 2017

Fourth Try: in tube SplintR Test with formamide[edit]

  • Try with 95C 3min denature before hybridization

Test Conditions[edit]

  1. Standard: SplintR only
  2. SplintR + 5% formamide
  3. SplintR + 7.5% formamide
  4. SplintR + 10% formamide
  5. SplintR + 12.5% formamide
  6. SplintR + 15% formamide
  7. SplintR + 17.5% formamide
  8. Positive Control: Ampligase
  • For each test conditions have
    • one sample with ALL padlock probes and template
      • Should see amplification
    • one sample with all padlock probes with NO MALAT1 template
      • Should not see amplification

Padlock Probes and Template[edit]

  • ppCUX2
  • ppBCL11B
  • ppRELN_1
  • ppGFAP
  • ppMALAT1
    • /5Phos/TTTCTGCCTTTACTTATCAATTCCTTCAGCTTCCCGATATCCGACGGTCTACTTCGTCGCGTCAGACCAAATGGAGGTATGACATATAATCT
  • Template for ppMALAT1: MALAT1_template
    • /5AmMC6/GAATTGATAAGTAAAGGCAGAAA AGATTATATGTCATACCTCCAT

Protocol[edit]

Sample # Condition 30nM PP + Template 10X Buffer Formamide H2O Total
1 SplintR 4.5 or 5.4 (0.9ul of each 1uM oligo) 3 0 22.5 or 21.6 30
2 SplintR + 5% formamide 4.5 or 5.4 (0.9ul of each 1uM oligo) 3 1.5 21 or 20.1 30
3 SplintR + 7.5% formamide 4.5 or 5.4 (0.9ul of each 1uM oligo) 3 2.25 20.25 or 19.35 30
4 SplintR + 10% formamide 4.5 or 5.4 (0.9ul of each 1uM oligo) 3 3 19.5 or 18.6 30
5 SplintR + 12.5% formamide 4.5 or 5.4 (0.9ul of each 1uM oligo) 3 3.75 18.75 or 17.85 30
6 SplintR + 15% formamide 4.5 or 5.4 (0.9ul of each 1uM oligo) 3 4.5 18 or 17.1 30
7 SplintR + 17.5 formamide 4.5 or 5.4 (0.9ul of each 1uM oligo) 3 5.25 17.25 or 16.35 30
8 Ampligase 4.5 or 5.4 (0.9ul of each 1uM oligo) 3 0 22.5 or 21.6 30
  1. Combine padlock probes and template in 1X Ligase buffer and possibly formamide
  2. Add mineral oil on top
  3. Incubate at 95C for 10min
  4. Incubate at 55C for 18hr
  5. To sample 8 add 3ul Ampligase Mix and incubate at 55C for 1hr30min
    • Ampligase Mix: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O
  6. Move samples 1-7 to 37C
  7. Add 3ul SplintR Mix and incubate 15min
    • SplintR Mix: 27ul SplintR + 4.5ul 10X SplintR Buffer + 13.5ul H2O
  8. Incubate at 94C for 10min
  9. Put all samples on ice and add 2ul Exo I/III mix
  10. Incubate at 37C for 1hr
  11. Incubate at 94C for 10min
  12. qPCR all 16 samples with triplicates
Components 1X Volume 48X Volume
Captured template 1 0
10uM ISB_CA_AF 0.4 19.2
10uM ISB_CA_AR.T2 0.4 19.2
2X KAPA SYBG MM 12.5 600
H2O 10.7 513.6
Total 25 1,152
  • Aliquot 24ul from 48X master mix and add 1ul captured template
 Program
 98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min

Results[edit]

raw data here
File:20170520 qPCR PPcapture SplintRformamide ScatterPlot.PNG

Conclusion[edit]

  • High temp denaturing beforehand definitely changed the results, theoretically removing the formamide's effect of denaturing secondary structures
  • Still similar trend that isn't 100% clear...
  • Need to repeat