Matt:LabNotes/2017-4-7

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SplintR RNA template specificity[edit]

Purpose[edit]

Samples[edit]

  1. PP + gDNA + SplintR
  2. PP + SplintR
  3. PP -ppCUX2 + SplintR
  4. PP (negative control for qPCR)
  5. PP + CircLigase II (positive control)

Protocol[edit]

  1. SplintR reaction for Samples 1-4
    • Incubate at 25C for 20min
    • Heat inactivate at 65C for 20min
Sample 10X SplintR Buffer SplintR Ligase 10uM ppBCL11B 10uM ppCUX2 10uM ppGFAP 10uM ppRELN_1 gDNA 12878 80.3ng/ul H2O Total
1 3 3 0.3 0.3 0.3 0.3 3 19.8 30
2 3 3 0.3 0.3 0.3 0.3 0 22.8 30
3 3 3 0.3 0 0.3 0.3 0 23.1 30
4 0 0 0.3 0.3 0.3 0.3 0 28.8 30
  1. Circligase II reaction with 400nM total
    • Incubate at 60C for 1hr
    • Inactive at 80C for 10min
Components Volume
CircLigase II 10X Buffer 3
CircLigase II Enzyme 1.5
5M Betaine 6
50mM MnCl2 1.5
10uM ppBCL11B 0.3
10uM ppCUX2 0.3
10uM ppGFAP 0.3
10uM ppRELN_1 0.3
H2O 16.8
Total 30
  1. Split each sample into 2 15ul samples
  2. Exo I/III digest one of each sample by adding 1ul Exo I and 1ul Exo III
    • Incubate at 37C for 1.5 hour
  3. Purify all 10 samples with Zymo ssDNA/RNA columns
    • Elute 15ul each
  4. qPCR all 10 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

File:20170408 SplintRNoTemplateTest qPCR.PNG

  1. Run gel on both
    1. PP + gDNA + SplintR
    2. PP + SplintR
    3. PP -ppCUX2 + SplintR
    4. PP (negative control for qPCR)
    5. PP + CircLigase II (positive control)
    6. PP + gDNA + SplintR (digested)
    7. PP + SplintR (digested)
    8. PP -ppCUX2 + SplintR (digested)
    9. PP (negative control for qPCR) (digested)
    10. PP + CircLigase II (positive control) (digested)

File:2017-04-08 SplintR DNAsplint Test.jpg

Conclusion[edit]

  • SplintR ligates padlock probes on DNA splints at slightly less efficiency than Circligase II
  • Negative control worked: linear padlock probes can not be amplified with these PCR primers
  • Exo I/III digestion improved efficiency of PCR of circular templates
  • SPECIFICITY MUST BE CONTROLLED WITH HIGH TEMP ANNEALING AND THOROUGH WASHING