Matt:LabNotes/2016-9-9: Difference between revisions

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[[Media:SplintR_Ligase_Test_qPCR_-_Enzyme_Digest.xlsx|Excel sheet with full PCR data]]<br>
[[Media:SplintR_Ligase_Test_qPCR_-_Enzyme_Digest.xlsx|Excel sheet with full PCR data]]<br>
[[File:SplintR_Ligase_Test_qPCR_-_Enzyme_Digest.JPG|450px]]
[[File:SplintR_Ligase_Test_qPCR_-_Enzyme_Digest.JPG|450px]]
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==Results & Conclusion==
*Pos Ctrl (V6 probes capture gDNA with Ampligase) has low amount of ligated padlock probes
**High Ct in PCR curve
**I guess 300ng gDNA has fewer targets than 30ng RNA
**But same low level as Neg Ctrl (Ampligase used on DNA/RNA hybrid)
*NTC (V6 captures '''nothing''' with SplintR) has same amount of ligated padlock probes as Experimental samples (V6 captures RNA with SplintR)
**suggests SplintR doesn't need RNA splint to ligate DNA
*CONCLUSION: Need to check literature and/or call NEB but it seems SplintR has high ssDNA ligase activity, making it unusable for high specificity padlock probe applications
**Checked and there is no reported ssDNA ligase activity, therefore it must be probes annealing to other probes
**"Template-independent ligation"
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Revision as of 21:57, 9 September 2016


SplintR Ligase Test 2

Reference

Experimental Outline

  1. V8 Padlock Probe capture to RNA
    • Ran out of V6 probes used last time
    • V8 is a subset of V6 probes (targets constitutive exons instead of contigs of exons)
  2. Quantify captured products by qPCR
    • Also adds Illumina sequencing adapters

Sample Groups

  • Use Universal Human Reference RNA (UHRR 740000-41)
  • 3 RNA template concentrations for each (30ng, 150ng and 820ng)
    • For DNA template positive control only do 1 sample using 300ng gDNA 12878
  1. NTC - 8.19ng V8 - SplintR
  2. NTC - 40.9ng V8 - SplintR
  3. NTC - 221.1ng V8 - SplintR
  4. 30ng RNA - 8.19ng V8 - SplintR
  5. 150ng RNA - 40.9ng V8 - SplintR
  6. 820ng RNA - 221.1ng V8 - SplintR
  7. PosCtrl: 300ng DNA - 16.38ng V8 - Ampligase
  8. NegCtrl: 150ng RNA - 40.9ng V8 - Ampligase
  • Summary:
    • 3 NTC samples with varying amount of padlock probes that matches experimental sample
    • 3 experimental samples of 30ng, 150ng, and 820ng UHRR
    • 1 PosCtrl that uses Ampligase for V8 to capture 300ng gDNA (876:1 probe:target ratio)
    • 1 NegCtrl that uses Ampligase for V8 to capture 150ng UHRR

Experiment

V8 Padlock Probe Capture

  • Dilute 1.5ul of 1ug/ul UHRR into 30ul total(50ng/ul final conc)
  • DNA is 12878 80.3ng/ul
  • V8 padlock probes: 874nM
    • Add 5.2ul to samples 3 & 6 and then dilute remaining ~4ul to 19ul final volume
Sample # RNA (50ng/ul) DNA (80.3ng/ul) V8 (42.7ng/ul) V8 (8.19ng/ul) 10X SplintR Buffer (*=Ampligase) H2O Total
1 0 0 0 1 3 26 30
2 0 0 0 5 3 22 30
3 0 0 5.2 0 3 21.8 30
4 0.6 0 0 1 3 25.4 30
5 3 0 0 5 3 19 30
6 16.4 0 5.2 0 3 5.4 30
7 0 3.8 0 2 3* 21.2 30
8 3 0 0 5 3* 19 30
  • Added 50ul mineral oil on top

Program

  • 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
  • Add 3ul Enzyme mix prepared on ice!
    • Samples 7-8: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O
    • Samples 1-6: 12ul SplintR + 2ul 10X SplintR Buffer + 6ul H2O
  • Incubate at 37C for 15min
  • 94C for 2min
  • Enzyme digest template 37C for 1 hr
    • 2ul Exo I/III (1:1) for DNA
    • 2ul RNaseH and Riboshredder (1:1) for RNA
      • First add 1ul 5M NaCl (100nM NaCl or KCl for Riboshredder)
  • 94C for 2min