Daniel:Notebook/ComboLock/2017-6-5

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Sensitivity and Specificity Experiments[edit]

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The purpose of this experiment will be to test the sensitivity and specificity of the ComboLock system using positive controls. The sample matrix is below, along with a column for rationales of why the sample exists in the first place. Note that


Sample No Template T1 C Probes T2 C Probes T1 Amt T2 Amt Test
1 T1 X   20 pmol 0 +++ Control
2 T2   X 0 20 pmol +++ Control-Does T2 work?
3 AA X   0 0 Negative Control
4 BB X   0 0 Negative Control
5 T1   X 20 pmol 0 Negative Control-Specificity
6 T2 X   0 20 pmol Negative Control-Specificity
7 T1/T2 50/50 X X 10 pmol 10 pmol Orthogonality
8 T1/T2 10/90 X X 2 pmol 18 pmol Sensitivity 10:1
9 T1/T2 1/99 X   200 fmol 19.8 pmol Sensitivity 100:1
10 T1/T2 1/9999 X   2 fmol 19.998 pmol Sensitivity 10K:1 (1000 copies of transcript in 100K cells)

Protocol[edit]

  1. Template dilutions
    1. Mix templates at appropriate ratios
      1. T1/T2 50/50: Mix 2 uL each oligo (10 uM each)
      2. T1/T2 10/90: Mix 4 uL each oligo (1 uM T1 and 10 uM T2)
      3. T1/T2 1/99: Mix 4 uL each oligo (100 nM T1 and 10 uM T2)
      4. T1/T2 1/9999: Mix 4 uL each oligo (1 nM T1 and 10 uM T2)
    2. To make dilutions, mix 5 uL sample in 45 uL nfH2O
    3. All samples are approximately 40 pmol added (2X bead loading)
    4. Pre-mix templates before bead binding
  2. Template-Bead Binding
    1. Suspend 2 uL (20 ug) beads per sample in 100 uL wash buffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Resuspend oligos in mixture of 20 uL Dynabuffer, 16 uL nfH2O
    4. 4 uL appropriate template oligo mix according to sample matrix(Total beads=20 ug=10 pmol available binding; 2X excess) 6X samples=12 uL
    5. Incubate at RT for 15 min
    6. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
  3. C Probe Hybridization
    1. Combine following into 0.2 mL tubes
    2. Reagent uL per Sample T1 Mix (6X) T2 Mix (2X) T1/T2 Mix (2X)
      PCCA-03 (10 uM) 1 6 0 2
      PCCB-03 (10 uM) 1 6 0 2
      PCCA-T2-03 (10 uM) 1 0 2 2
      PCCB-T2-03 (10 uM) 1 0 2 2
      DynaBeads Wash Buffer 10 60 20 20
      nfH2O 8 48 16 12
      Total 20 120 40 40
    3. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    4. Add 20 uL appropriate probe mixture to beads following sample matrix above
    5. Incubate at 40C for 1 hour with agitation
    6. Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
    7. Repeat wash step above
    8. Repeat wash step above

Protocol-Part 2[edit]

  1. Lock and Latch Hybridization/Ligation
    1. Set up the following master mix
    2. Reagent Stock Conc Final Conc./Amt uL added Master Mix (10.2X)
      T4 Ligase Reaction Buffer 5X 1X 4 40.8
      Latch Oligo 10 uM 10 pmol 1 10.2
      Bolt Oligo 10 uM 10 pmol 1 10.2
      nfH2O NA NA 13 132.6
      Total     19 193.8
    3. Pre-anneal latch/bolt
      1. Heat mix to 95C for 3 minutes
      2. Lower mix to 55C at rate of 0.2C/sec
      3. Incubate at 55C for 10 minutes
    4. Add 19 uL annealed probe mix to each sample
    5. Incubate at 55C for 10 minutes
    6. Incubate at 25C for 10 minutes
    7. Lower the temperature to 20C; wait 30 seconds then take off incubator
    8. Add 1 uL T4 Ligase to each reaction
    9. Incubate at RT for 15 min
    10. Heat kill enzyme by incubating at 65C for 10 minutes
    11. Wash beads with 100 uL Dynabuffer
    ******
    
  2. Padlock Hybridization
    1. Add 20 uL of following 10.2X Master Mix:
      1. 10.2 uL 10 uM Padlock0601
      2. 20.4 uL 10X Amp Ligase buffer
      3. 173.4 uL nfH2O
    2. Incubate at 70C for 5 min
    3. Incubate at 50C for 15 minutes
    4. Wash once with wash buffer
    5. Resuspend in 20 uL 1X Amp Ligase buffer
    ******
    
  3. Circularization
    1. Make the following master mix
    2. Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (10.5x) (uL)
      NAD+ 5 mM 40 nmol 8 84
      dNTP 1 mM 600 pmol 0.6 6.3
      Betaine 5 M 15 umol 3 31.5
      10X AmpLigase Buffer 10X 1X 4 42
      Amp Ligase 5 U/uL 10 U 2 21
      Phusion HF DNA Polymerase 2000 U/mL 6.4U 3.2 33.6
      nf H2O     19.2 201.6
      Total     40 420
    3. Add 40 uL circularization mix to each sample
    4. Incubate at 55C for 4 hours
    5. Incubate at 95C for 5 minutes
    6. Hold at 16C


Continued tomorrow