Matt:LabNotes/2017-4-22

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In tube SplintR Test with formamide and ET SSB[edit]

Test Conditions[edit]

  1. Standard: SplintR only
  2. SplintR incubated for 30min instead of 15min
  3. SplintR + 10% formamide
  4. SplintR + 20% formamide
  5. SplintR + 30% formamide
  6. SplintR + 250ng ET SSB
  7. SplintR + 250ng ET SSB added first
  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 and template MINUS ppMALAT1
      • Should not see amplification

Padlock Probes and Template[edit]

  • Same as Matt:LabNotes/2017-4-7 with the addition of ppMALAT1 for consistency
  • ppCUX2
  • ppBCL11B
  • ppRELN_1
  • ppGFAP
  • ppMALAT1
    • /5Phos/TTTCTGCCTTTACTTATCAATTCCTTCAGCTTCCCGATATCCGACGGTCTACTTCGTCGCGTCAGACCAAATGGAGGTATGACATATAATCT
  • Template for ppMALAT1: MALAT1_template
    • /5AmMC6/GAATTGATAAGTAAAGGCAGAAA AGATTATATGTCATACCTCCAT

Protocol[edit]

Sample # Condition 100nM template + PP mix 55C 17hr 55C 37C Exo I/III
1 SplintR 15min 1.5 or 1.8ul (0.3ul of each 10uM oligo) 3ul SplintR Buffer + H2O to 30ul - Add 3ul SplintR Mix 1ul Exo I + 1ul Exo II + 0.4ul ET SSB
2 SplintR 30min 1.5 or 1.8ul (0.3ul of each 10uM oligo) 3ul SplintR Buffer + H2O to 30ul - Add 3ul SplintR Mix 1ul Exo I + 1ul Exo II + 0.4ul ET SSB
3 SplintR + 10% formamide 1.5 or 1.8ul (0.3ul of each 10uM oligo) 3ul SplintR Buffer + 3ul formamide + H2O to 30ul - Add 3ul SplintR Mix 1ul Exo I + 1ul Exo II + 0.4ul ET SSB
4 SplintR + 20% formamide 1.5 or 1.8ul (0.3ul of each 10uM oligo) 3ul SplintR Buffer + 6ul formamide + H2O to 30ul - Add 3ul SplintR Mix 1ul Exo I + 1ul Exo II + 0.4ul ET SSB
5 SplintR + 30% formamide 1.5 or 1.8ul (0.3ul of each 10uM oligo) 3ul SplintR Buffer + 9ul formamide + H2O to 30ul - Add 3ul SplintR Mix 1ul Exo I + 1ul Exo II + 0.4ul ET SSB
6 SplintR + ET SSB 1.5 or 1.8ul (0.3ul of each 10uM oligo) 3ul SplintR Buffer + H2O to 30ul - Add 3ul SplintR Mix + 0.4ul ET SSB 1ul Exo I + 1ul Exo II
7 SplintR + 2step ET SSB 1.5 or 1.8ul (0.3ul of each 10uM oligo) 3ul SplintR Buffer + H2O to 30ul Add 0.4ul ET SSB Add 3ul SplintR Mix 1ul Exo I + 1ul Exo II
8 Ampligase 1.5 or 1.8ul (0.3ul of each 10uM oligo) 3ul Ampligase Buffer + H2O to 30ul Add 3ul Ampligase Mix - 1ul Exo I + 1ul Exo II + 0.4ul ET SSB
  1. Combine padlock probes and template in 1X Ligase buffer and possibly formamide
  2. Add mineral oil on top
  3. Incubate at 55C for 17hr
  4. To sample 7 (2-step ET SSB) add 200ng (0.4ul) of ET SSB and keep at 55C for 30min
  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. After sample 7 has ET SSB for 30min 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
    • 30min incubation for sample 2
    • Also add 0.4ul ET SSB to sample 6
  8. Put all samples on ice and add 2ul Exo I/III mix
    • Also add 0.4ul ET SSB to samples 1-5 and 8
  9. Incubate at 37C for 1hr
  10. 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

Mistakes[edit]

  • Forgot to heat inactivate (incubate at 94C 10min) after ligation and before Exo I/III
  • Accidentally added 3ul 10X SplintR Buffer to samples 1-3 that HAVE ppMALAT1
    • Still added 3ul SplintR Mix afterwards
  • Do zymo columns after Exo I/III next time

Results[edit]

raw data here
File:20170423 qPCR ppCapture SplintRformamideETSSB.PNG

  • Possible options: 10% formamide

File:20170423 qPCR PPcapture SplintRformamideETSSB ScatterPlot.PNG

  • No difference between 15min and 30min SplintR incubation times
  • ET SSB added does increase the number of captured padlock probes
    • Adding ET SSB before SplintR vs during SplintR makes no difference
  • Next time do a ET SSB + 10% formamide sample
  • Next time try less template concentration... see if it can distinguish the smaller amount