Daniel:Notebook/ComboLock/2016-10-17: Difference between revisions

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File:20161017-qPCR-Padlock0301-Dilutions2.png|qPCR image
File:20161017-qPCR-Padlock0301-Dilutions2.png|qPCR image
File:|Gel image 1
File:2016-10-17-Padlock0301-Dilutions2-Gel1.png|Gel image 1
File:|Gel image 2
File:2016-10-17-Padlock0301-Dilutions2-Gel2.png|Gel image 2
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Revision as of 20:07, 17 October 2016

BSA Ab-Oligo Linkage (From Friday)

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Friday I accidentally let the post-sulfo SMCC reaction elute into the wash container, which probably messed things up. So I'm repeating that part of the experiment and will mix the new Ab fraction with the oligo fraction prepared on Friday.

Protocol

  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
  4. Ab-Oligo Mixing
    1. Mix SMCC-treated antibodies with DTT-treated oligonucleotides at 10X molar excess of oligo to Ab
    2. Transfer to pre-wet Slide-A-Lyzer Mini 7 MWCO dialysis cup
    3. Dialyze in 1 L PBS with 5 mM EDTA at 4C for 2 days with one buffer exchange to PBS
    4. Dilute probes to 75 ug/mL in PBS buffer with 4 mM EDTA, 35 ug/mL ssDNA, 0.1% fish gelatin, and 20 mM Tris HCl
    5. Store at 4C

Padlock0301 Dilutions Test (Started Thursday 10-13)

I'm going to repeat the PCR test, just in case.

  1. 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
  2. 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

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

Cell Trial