Daniel:Notebook/ComboLock/2016-10-14

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Revision as of 15:47, 20 October 2016 by >Djacobse (→‎Cell Preparation)
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Padlock0301 Dilution Test (Started yesterday)[edit]

Back to Calendar

Protocol[edit]

  1. Circularization
    1. Heat inactivate enzyme by incubating for 2 minutes at 94C
  2. Exonuclease Digestion
    1. Prepare exonuclease I/III mix by mixing 32.5 uL exonuclease I (20 units/μL) and 6.5 uL exonuclease III (100 units/μL) in 1:1 ratio
    2. Add 2 μL of exonuclease I/III mix to reaction
    3. Mix the reaction by swirling pipette around the well 5 times
    4. Incubate reaction at 37 ºC for 2 hours
    5. Heat inactivate enzyme by incubating at 94C for 5 minutes
  3. qPCR
    1. Prepare the following master mix
      1. 334.25 uL nfH2O
      2. 9.55 uL 100 uM AmpF-CLv2
      3. 477.5 uL Kapa SYBR Fast MM
    2. Add 43 uL master mix to each well
    3. Add sample and AmpR indexes according to sample table in the next section
    4. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x24
      7. 72C 2 min
      8. 16C hold
  4. TBE Gel
    1. Mix 176 uL TBE, 44 uL 6x loading dye
    2. Aliquot 10 uL per sample/ladder lane onto parafilm
    3. Add 2 uL of sample or ladder to correct drop
    4. Load 10 uL in to well
    5. Run gel for 22 minutes at 250V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc

qPCR Sample Table[edit]

Sample Template Molecule Count Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 100 uM AmpF-CLv2 10 uM AmpR6.3-IndXX H2O Total Volume (uL)
Sample 6A 10 amol A1 20 2 25 0.5 5 17.5 50
Sample 6B 10 amol B1 20 2 25 0.5 5 17.5 50
Sample 6C 10 amol C1 20 2 25 0.5 5 17.5 50
Sample 8A 100 zmol D1 21 2 25 0.5 5 17.5 50
Sample 8B 100 zmol E1 21 2 25 0.5 5 17.5 50
Sample 8C 100 zmol F1 21 2 25 0.5 5 17.5 50
Sample 9A 10 zmol G1 22 2 25 0.5 5 17.5 50
Sample 9B 10 zmol H1 22 2 25 0.5 5 17.5 50
Sample 9C 10 zmol A12 22 2 25 0.5 5 17.5 50
Sample 10A 1 zmol B12 23 2 25 0.5 5 17.5 50
Sample 10B 1 zmol C12 23 2 25 0.5 5 17.5 50
Sample 10C 1 zmol D12 23 2 25 0.5 5 17.5 50
Sample 11A 100 ymol E12 24 2 25 0.5 5 17.5 50
Sample 11B 100 ymol F12 24 2 25 0.5 5 17.5 50
Sample 11C 100 ymol G12 24 2 25 0.5 5 17.5 50
Sample 12A 10 ymol H12 25 2 25 0.5 5 17.5 50
Sample 12B 10 ymol A2 25 2 25 0.5 5 17.5 50
Sample 12C 10 ymol B2 25 2 25 0.5 5 17.5 50
NTC 0 C2 26 2 25 0.5 5 19.5 50

Results[edit]

BSA Ab-Oligo Linkage[edit]

  1. Buffer Prep
    1. Mix 1.4 mL of monobasic and 3.6 mL of dibasic and 45 mL ddH2O to make 50 mL of 100 mM (buffer pH 7.2)
  2. Antibody Activation
    1. Resuspend antibodies at 1 mg/mL in PBS
    2. Equilibrate a 40 kDa spin column (or plate) 4 times with 100 mM phosphate buffer, pH 7.3; 250 uL, spin at 1000xg for 2 min
    3. Add 20 uL antibody to the column and spin for 3 min at 1000xg; collect in a new plate
    4. Dissolve sulfo-SMCC to 3.33 mM in 100mM phosphate buffer
    5. Add 2 uL sulfo-SMCC to sample and incubate for 2 hr at 4C with 3 times intermittent mixing
    6. Equilibrate a new 40 kDa column with 100 mM phosphate buffer with 20 mM EDTA
    7. Transfer antibodies to new column and spin for 3 min at 1000xg
  3. Oligonucleotide Activation
    1. Resuspend oligonucleotide in 1 mM in 100 mM phosphate buffer with 20 mM EDTA
    2. Add 1.3 uL oligonucleotide to tube (or plate)
    3. Add 2.2 uL 40 mM DTT; incubate at 95C for 2 min followed by 1 hr at 37C
    4. Add 20 uL PBS with 20 mM EDTA
    5. Remove excess DTT using two consecutive Zeba 7 kDa columns equilibrated with 100 mM PBS

I accidentally eluted the Ab fraction into a wash collector (with wash buffer in it), so I'm going to have to repeat the experiment on Monday

Cell Preparation[edit]

Prepping U87-MG cells for an experiment. Since I have a potential cold and I want to nurse it, I'll try and do the actual experiment on Monday.

  1. Fix cells
    1. Pellet cells at 600g for 3 min
    2. Fix cells in 5 mL EMEM with 1.6% paraformaldehyde for 10 min at RT
    3. Wash cells with wash buffer
    4. Pellet cells and permeabilize with 2 mL ice-cold methanol for 10 min on ice; split cells into individual 1.5 mL tubes
    5. Store cells in -80C