Daniel:Notebook/ComboLock/2017-3-21

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Circularization Comparison (T4 Poly/Ligase; Started Wednesday)[edit]

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Bgl Digestion[edit]

Because I CAN. Also, will determine if there is an improvement in signal post-digestion.

Sample Matrix

Sample Origin Condition Sample Numbers
F T4 Ligase 1,4
G Phusion/Amp (Unpurified) 1,4
H Phusion/AmpHS (Unpurified) 1,4
  1. Bglii Digestion
    1. Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest
    2. Make the following 4.2X master mix
    3. Reagent Single Rxn Vol 14.2X MM uL
      RCA Reaction Product 5 0
      Bglii Cutter Primer (10 uM) 5 71
      10X Buffer 3.1 2 28.4
      Bglii 1 0
      nfH2O 7 99.4
      Total 20 198.8
    4. Aliquot 14 uL master mix into new tubes
    5. Add 5 uL appropriate sample to each tube
    6. Use the following thermocycler program
      1. 5 min 95C
      2. 10 min 50C
      3. 1 hr 37C; When this cycle starts add 1 uL Bglii
      4. Heat kill with 65C for 20 min
  2. qPCR
    1. Make qPCR master mix according to following recipe
      1. 319.2 uL nfH2O
      2. 15.2 uL AmpF
      3. 15.2 uL AmpR-Ind22
      4. 380 uL Kapa SYBR Fast 2X Master Mix
    2. Add 48 uL master mix to each well
    3. Add 2 uL sample according to plate layout
    4. File:PlateLayout-T4Circ-BgliiDigest-20170321.png
    5. 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 120 uL TBE and 40 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

Results[edit]