Matt:LabNotes/2017-4-22

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Revision as of 23:11, 23 April 2017 by >Mzcai
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In tube SplintR Test with formamide and ET SSB

Test Conditions

  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

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

Protocol

  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
  1. To sample 7 (2-step ET SSB) add 200ng (0.4ul) of ET SSB and keep at 55C for 30min
  2. To sample 8 add 3ul Ampligase Mix and incubate at 55C for 1hr30min
    • Ampligase Mix: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O
  3. After sample 7 has ET SSB for 30min move samples 1-7 to 37C
  4. 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
  5. Put all samples on ice and add 2ul Exo I/III mix
    • Also add 0.4ul ET SSB to samples 1-5 and 8
  6. Incubate at 37C for 1hr
  7. 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

  • 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