Daniel:Notebook/ComboLock/2017-3-16

From ZhangLabWiki
Jump to navigation Jump to search

Circularization Comparison (T4 Poly/Ligase; Started Yesterday)

Back to Calendar

Protocol-Part 4

  1. Exonuclease Digestion
    1. Mix 25 uL Exo I (20U/uL) and 5 uL ExoIII (100U/uL)
    2. Add 2 uL to each sample, mix by swirling pipette tip
    3. Incubate for 1.5 hours at 37C
    4. Heat kill by incubating for 5 min at 95C
  2. qPCR
    1. Make qPCR master mixes according to following recipe
    2. Reagent Single Rxn MMProd (15.2X) MM+ (C1, 4.2X)
      Primer Pair (fwd/rev) NA primer12*/AmpR-Ind21 primer24/primer4*
      Forward Primer (10 uM) 1 15.2 4.2
      Reverse Primer (10 uM) 1 15.2 4.2
      nfH2O 21 319.2 88.2
      2X Kapa SYBR Fast 25 380 105
      Total 48 729.6 201.6
    3. Add 48 uL master mix to each well
    4. Add 2 uL sample according to plate layout
    5. File:PlateLayout-20170316-T4Circ-PreRCATest.png
    6. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x30
      7. 72C 2 min
      8. 16C hold
  3. TBE Gel
    1. Mix 200 uL TBE and 50 uL 6X dye
    2. Aliquot 10 uL per sample on to parafilm
    3. Add 2 uL sample or 1.5 uL ladder to aliquots
    4. Mix and add 10 uL per lane to gel
    5. Run gel at 230V for 24 minutes
    6. Add 2 uL SYBR gold and incubate on shaker for 3 minutes
    7. Rinse and image in gel doc

Pre-RCA Results

Protocol-Part 3-Y Samples

  1. Padlock Hybridization-Y Samples
    1. Bake samples at 95 C and vortex to resuspend
    2. Make the following master mix
      1. 4.2 uL Padlock0601 (10 uM)
      2. 8.4 uL Amp Ligase 10X buffer
      3. 50.4 uL nfH2O
    3. Add 15 uL master mix to appropriate tubes
    4. Add 5 uL sample according to yesterday's tables
    5. Incubate at 50C for 20 minutes
    ******
    
  2. Circularization
    1. Make the following master mix
    2. Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (4.5x) (uL)
      NAD+ 5 mM 40 nmol 8 36
      dNTP 1 mM 600 pmol 0.6 2.7
      Betaine 5 M 15 umol 3 13.5
      10X AmpLigase Buffer 10X 1X 2 9
      Amp Ligase 5 U/uL 10 U 2 9
      Phusion HF DNA Polymerase 2000 U/mL 6.4U 3.2 14.4
      nf H2O     1.2 5.4
      Total     20 90
    3. Follow the workflow in yesterday's diagrams
    ******
    
  3. Exonuclease Digestion
    1. Mix 7.5 uL Exo I (20U/uL) and 1.5 uL ExoIII (100U/uL)
    2. Add 2 uL to each sample, mix by swirling pipette tip
    3. Incubate for 1.5 hours at 37C
    4. Heat kill by incubating for 5 min at 95C

Protocol-Part 5

  1. Column Purification-Qiaquick-Do this for 20 uL from Y and Z samples
    1. Add 100 uL (5X) PB (binding buffer) to 20 uL sample
    2. Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
    3. Add 750 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through;
    4. Repeat wash step;
    5. Dry spin column for 1 minute at 14000 rpm; discard flow through
    6. Let stand with cover open in fume hood for ~5 minutes
    7. Transfer column to a new 1.5 mL eppendorf tube
    8. Elute with 30 uL EB
    9. Let stand 1 minute
    10. Spin for 1 minute at 14000 rpm
    Sample Origin Sample New Letter
    Y P
    Z Q
    ******
    
  2. Rolling Circle Amplification
  3. Sample Conversion Table
    Sample Origin Sample New Letter Sample Numbers
    X F 1-5
    Y G 1,4
    Z H 1,4
    P I 1,4
    Q K 1,4
    1. Prepare 8.2X Master mix according to table below
    2. Add 12.5 uL master mix to each tube
    3. Add 4 uL template according to table
    4. Add 2.5 uL correct primer according to table
    5. Reagent Single Rxn Vol 13.2X (A) MM uL 13.2X (B) MM uL
      Template 4 0 0
      RCA Primer (10 uM) 2.5 33 0
      RCA Phosphothiorate Primer (10 uM) 2.5 0 33
      dNTP (1 mM) 5 66 66
      10X Buffer 2 26.4 26.4
      Phi29 1 13.2 13.2
      BSA (10 mg/mL) 0.4 5.28 5.28
      nfH2O 5.1 67.32 67.32
      Total 20 211.2 211.2
    6. Incubate at 95 C for 5 minutes
    7. Lower to 55C for 15 minutes
    8. Lower to 30C for at least 2 minutes
    9. Add 1 uL Phi29 polymerase to each sample
    10. Incubate 3 hours at 30C
    11. Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C