Matt:LabNotes/2017-5-8: Difference between revisions

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==Repeat qPCR==
==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
  98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x30 -> 72C 3min
==Zymo column purify and Repeat qPCR==
#Purify ssDNA from 20ul of 16 samples
#Elute 10ul each
#qPCR all 16 samples 1ul each with triplicates
#qPCR all 16 samples 1ul each with triplicates



Revision as of 18:39, 16 May 2017

Second Try: in tube SplintR Test with formamide and ET SSB

Test Conditions

  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 + 10% formamide + 250ng ET SSB
  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

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

Protocol

Sample # Condition 10nM PP + Template 10X Buffer Formamide H2O Total
1 SplintR 1.5 or 1.8 (0.3ul of each 1uM oligo) 3 0 25.5 or 25.2 30
2 SplintR + 5% formamide 1.5 or 1.8 (0.3ul of each 1uM oligo) 3 1.5 24 or 23.7 30
3 SplintR + 7.5% formamide 1.5 or 1.8 (0.3ul of each 1uM oligo) 3 2.25 23.25 or 22.95 30
4 SplintR + 10% formamide 1.5 or 1.8 (0.3ul of each 1uM oligo) 3 3 22.5 or 22.2 30
5 SplintR + 12.5% formamide 1.5 or 1.8 (0.3ul of each 1uM oligo) 3 3.75 21.75 or 21.45 30
6 SplintR + 15% formamide 1.5 or 1.8 (0.3ul of each 1uM oligo) 3 4.5 21 or 20.7 30
7 SplintR + 10% formamide + ET SSB 1.5 or 1.8 (0.3ul of each 1uM oligo) 3 3 22.5 or 22.2 30
8 Ampligase 1.5 or 1.8 (0.3ul of each 1uM oligo) 3 0 25.5 or 25.2 30
  1. Combine padlock probes and template in 1X Ligase buffer and possibly formamide
  2. Add mineral oil on top
  3. Incubate at 55C for 18hr
  4. To sample 8 add 3ul Ampligase Mix and incubate at 55C for 1hr30min
    • Ampligase Mix: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O
  5. Move samples 1-7 to 37C
  6. Add 3ul SplintR Mix and incubate 15min
    • SplintR Mix: 27ul SplintR + 4.5ul 10X SplintR Buffer + 13.5ul H2O
    • Also add 0.4ul ET SSB to sample 7
  7. Incubate at 94C for 10min
  8. Put all samples on ice and add 2ul Exo I/III mix
  9. Incubate at 37C for 1.5hr
  10. Incubate at 94C for 10min
  11. qPCR all 16 samples
Components 1X Volume 16X Volume
Captured template 5 0
10uM ISB_CA_AF 0.4 6.4
10uM ISB_CA_AR.T1 0.4 6.4
2X KAPA SYBG MM 12.5 200
H2O 6.7 107.2
Total 25 320
  • Aliquot 20ul from 16X master mix and add 5ul captured template
 Program
 98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min

Results

raw data here
File:20170510 qPCR PPcapture SplintRformamideETSSB.PNG

  • Only Ampligase stands out from the rest

File:20170510 qPCR PPcapture SplintRformamideETSSB ScatterPlot.PNG

  • 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

  1. qPCR all 16 samples 1ul each 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)x30 -> 72C 3min


Zymo column purify and Repeat qPCR

  1. Purify ssDNA from 20ul of 16 samples
  2. Elute 10ul each
  3. qPCR all 16 samples 1ul each 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)x30 -> 72C 3min