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

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>Mzcai
(Created page with "=Second Try: in tube SplintR Test with formamide and ET SSB= *[[Matt:LabNotes/2017-4-22|First try showed SplintR with 10% formamide had the best sensitivity and specificity of...")
 
>Mzcai
Line 36: Line 36:
| width="51" height="14" | Sample #
| width="51" height="14" | Sample #
| width="96" | Condition
| width="96" | Condition
| width="51" | 100nM PP + Template
| width="51" | 10nM PP + Template
| width="51" | 10X Buffer
| width="51" | 10X Buffer
| width="51" | Formamide
| width="51" | Formamide
Line 45: Line 45:
| align="right" height="14" | 1
| align="right" height="14" | 1
  | SplintR
  | SplintR
  | 1.5 or 1.8 (0.3ul of each 10nM oligo)
  | 1.5 or 1.8 (0.3ul of each 1uM oligo)
| align="right" | 3
| align="right" | 3
| align="right" | 0
| align="right" | 0
Line 54: Line 54:
| align="right" height="14" | 2
| align="right" height="14" | 2
  | SplintR + 5% formamide
  | SplintR + 5% formamide
  | 1.5 or 1.8 (0.3ul of each 10nM oligo)
  | 1.5 or 1.8 (0.3ul of each 1uM oligo)
| align="right" | 3
| align="right" | 3
| align="right" | 1.5
| align="right" | 1.5
Line 63: Line 63:
| align="right" height="14" | 3
| align="right" height="14" | 3
  | SplintR + 7.5% formamide
  | SplintR + 7.5% formamide
  | 1.5 or 1.8 (0.3ul of each 10nM oligo)
  | 1.5 or 1.8 (0.3ul of each 1uM oligo)
| align="right" | 3
| align="right" | 3
| align="right" | 2.25
| align="right" | 2.25
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| align="right" height="14" | 4
| align="right" height="14" | 4
  | SplintR + 10% formamide
  | SplintR + 10% formamide
  | 1.5 or 1.8 (0.3ul of each 10nM oligo)
  | 1.5 or 1.8 (0.3ul of each 1uM oligo)
| align="right" | 3
| align="right" | 3
| align="right" | 3
| align="right" | 3
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| align="right" height="14" | 5
| align="right" height="14" | 5
  | SplintR + 12.5% formamide
  | SplintR + 12.5% formamide
  | 1.5 or 1.8 (0.3ul of each 10nM oligo)
  | 1.5 or 1.8 (0.3ul of each 1uM oligo)
| align="right" | 3
| align="right" | 3
| align="right" | 3.75
| align="right" | 3.75
Line 90: Line 90:
| align="right" height="14" | 6
| align="right" height="14" | 6
  | SplintR + 15% formamide
  | SplintR + 15% formamide
  | 1.5 or 1.8 (0.3ul of each 10nM oligo)
  | 1.5 or 1.8 (0.3ul of each 1uM oligo)
| align="right" | 3
| align="right" | 3
| align="right" | 4.5
| align="right" | 4.5
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| align="right" height="14" | 7
| align="right" height="14" | 7
  | SplintR + 10% formamide + ET SSB
  | SplintR + 10% formamide + ET SSB
  | 1.5 or 1.8 (0.3ul of each 10nM oligo)
  | 1.5 or 1.8 (0.3ul of each 1uM oligo)
| align="right" | 3
| align="right" | 3
| align="right" | 3
| align="right" | 3
Line 108: Line 108:
| align="right" height="14" | 8
| align="right" height="14" | 8
  | Ampligase
  | Ampligase
  | 1.5 or 1.8 (0.3ul of each 10nM oligo)
  | 1.5 or 1.8 (0.3ul of each 1uM oligo)
| align="right" | 3
| align="right" | 3
| align="right" | 0
| align="right" | 0

Revision as of 01:53, 9 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 17hr