Daniel:Notebook/ComboLock/2017-3-15

From ZhangLabWiki
Revision as of 19:44, 16 March 2017 by >Djacobse (→‎Protocol-Part 3)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search

Circularization Comparison (T4 Poly/Ligase)[edit]

Back to Calendar

Based on the March 8 experiment I will try the protocol again with a special note for testing against the bolt oligo and paying close attention to circularization.

Protocol[edit]

Sample Matrix

Sample Condition
Sample 1 (AB) Normal
Sample 2 (AB) No Template
Sample 3 (AB) No C probes
Sample 4 (AB) Dephosphorylated
Sample 5 (AB) Positive Control (C4-C2)
  • Note:Because sample 5 is the finished product (ligated C probes), it will be started at step 5
******
  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 0.2 mL tubes
    2. Reagent uL per Sample Exp uL Total (1,2,4) No C uL Total (3)
      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; Start step 3 during this process
    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. Dephosphorylation (Bolt Oligo)
    1. Mix the following reaction
      1. 5 uL Bolt0301 (10 uM)
      2. 2 uL CutSmart Buffer (10X)
      3. 2 uL rSAP enzyme
      4. 16 uL nfH2O
    2. Incubate at 37C for 30 minutes
    3. Incubate at 65 for 5 minutes
    ******
    
  2. Lock and Latch Hybridization/Ligation
    1. Set up the following master mix
    2. Reagent Stock Conc Final Conc./Amt uL added Master Mix (6.2X) Master Mix NP (2.2X)
      T4 Ligase Reaction Buffer 5X 1X 4 24.8 8.8
      Latch Oligo 10 uM 10 pmol 1 6.2 2.2
      Bolt Oligo 10 uM 10 pmol 1 6.2 0
      Dephosphorylated Bolt 2.5 uM 10 pmol 8 0 17.6
      nfH2O NA NA 13 80.6 13.2
      Total     19 117.8 41.8
    3. Add 19 uL probe mix to each sample
    4. Incubate at 55C for 10 minutes
    5. Incubate at 25C for 10 minutes
    6. Lower the temperature to 20C; wait 30 seconds then take off incubator
    7. Add 1 uL T4 Ligase to each reaction
    8. Incubate at RT for 15 min
    9. Heat kill enzyme by incubating at 65C for 10 minutes

    Protocol-Part 3[edit]

    Sample Matrix 2-Circularization

    Sample Code Condition Samples Included (#)
    X T4 Polymerase & Ligase 1-5 (10)
    Y Phusion + Amp Ligase 1,4 (4)
    Z Phusion + Amp Ligase-Hot Start 1,4 (4)


    1. Padlock Hybridization
      1. Wash samples one time and resuspend in 30 uL nfH2O
      2. Bake samples at 95 C and vortex to resuspend
        • Note: Here I accidentally added the Phusion mix to the Y samples before adding the enzymes, so this is adjusted to only analyze the Z samples. Y samples will be processed tomorrow.
      3. Make the following master mixes
      4. Reagent MMX Unit MMZ Unit MMX (10.2X) MMZ (4.2X)
        Padlock0601 (10 uM) 1 1 10.2 4.2
        Sample 5 5 0 0
        Amp Ligase Buffer (10X) 0 2 0 8.4
        5X T4 Ligase Buffer 4 0 40.8 0
        dNTPs (1 mM) 2 0 20.4 0
        nfH2O 7 12 71.4 50.4
        Total 19 20 142.8 63
      5. Add 14 (X) or 15 (YZ) uL master mix to appropriate tubes
      6. Add 5 uL sample according to tables
      7. Incubate at 50C for 20 minutes
      ******
      
    2. Circularization
      1. For phusion samples, make the following master 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. Follow the workflow in the below diagrams
      4. File:CircProtocol-T4Poly.png Continued tomorrow