Daniel:Notebook/ComboLock/2017-3-1

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Padlock0601 Test[edit]

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Testing the new arrival of padlock0601, which also contains a restriction enzyme cutting site (Bam)

Protocol-Part 1[edit]

Sample Matrix

Sample Condition
Sample 1 (AB) Normal
Sample 2 (AB) No Template
Sample 3 (AB) No C probes
Sample 4 (AB) No phosphate on padlock (Padlock0501)
  1. Template-Bead Binding
    1. Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min
    4. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
  2. C Probe Hybridization
    1. Combine following into a 0.2 mL tube
    2. Reagent uL per Sample uL Total (Samples 1-2,4) uL Total (No C Probe)
      PCCA-03 (10 uM) 1 6 0
      PCCB-03 (10 uM) 1 6 0
      Wash buffer 3 18 10
      Total 5 30 10
    3. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    4. Add 5 uL probe mixture to beads
    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. Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant

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 (8.2X)
      T4 Ligase Reaction Buffer 5X 1X 4 32.8
      Latch Oligo 10 uM 10 pmol 1 8.2
      Lock Oligo 10 uM 10 pmol 1 8.2
      nfH2O NA NA 13 106.6
      Total     19 155.8
    3. Add 19 uL probe mix to each sample
    4. Incubate at 55C for 20 minutes
    5. Lower the temperature to 20C; wait 30 seconds then take off incubator
    6. Add 1 uL T4 Ligase to each reaction
    7. Incubate at RT for 15 min
    8. Heat kill enzyme by incubating at 65C for 10 minutes
    9. Wash twice with 100 uL wash buffer
  2. Padlock Hybridization
    1. Make the following master mixes
    2. Reagent 1X MMX MM-NP
      Padlock0501 (10 uM) 2 0 4.2
      Padlock0601 (10 uM) 2 12.2 0
      Amp Ligase Buffer (10X) 2 12.2 4.2
      nfH2O 16 97.6 33.6
      Total 20 122 42
    3. Resuspend samples 1-3 in 20 uL MMX and samples 4A,4B in 20 uL MMNP
    4. Incubate at 50C for 30 minutes
  3. Circularization
    1. Prepare 8.5X uL Phusion mix
    2. Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (8.5x) (uL)
      NAD+ 5 mM 40 nmol 8 68
      dNTP 1 mM 600 pmol 0.6 5.1
      Betaine 5 M 15 umol 3 25.5
      10X AmpLigase Buffer 10X 1X 2 17
      Amp Ligase 5 U/uL 10 U 2 17
      Phusion HF DNA Polymerase 2000 U/mL 6.4U 3.2 27.2
      nf H2O     1.2 8.4
      Total     20 140
    3. Add 20 uL phusion mix to 20 uL reaction
    4. Incubate at 55C for 2 hours

Continued tomorrow