Daniel:Notebook/ComboLock/2016-5-26: Difference between revisions

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<li>For VIM-PA and VIM-PB:</li>
<li>For VIM-PA and VIM-PB:</li>
<ol type="a">
<ol type="a">
<li>Add 1.3 uL 100 uM oligonucleotide to tube</li>
<li>Add 13 uL 100 uM oligonucleotide to tube</li>
<li>Add 2.2 uL 40 mM DTT</li>
<li>Add 22 uL 40 mM DTT</li>
<li>Incubate at 95C for 2 min followed by 1 hr at 37C</li>
<li>Incubate at 95C for 2 min followed by 1 hr at 37C</li>
<li>Add 20 uL PBS with 20 mM EDTA</li>
<li>Add 165 uL PBS with 20 mM EDTA</li>
<li>Remove excess DTT  using two consecutive [[Media:Zeba_Spin_Desalt_Colomn_7K_MWCO_UG_Manual.pdf|Zeba 7 kDa]] columns equilibrated with 100 mM PBS</li>
<li>Remove excess DTT  using two consecutive [[Media:Zeba_Spin_Desalt_Colomn_7K_MWCO_UG_Manual.pdf|Zeba 7 kDa]] columns equilibrated with 100 mM PBS (10X PBS)</li>
<ol type="a"><li>Spin at 1500xg for 1 minute for equilibrium; 2 min for sample</li></ol>
<ol type="a"><li>Spin at 1500xg for 1 minute for equilibrium; 2 min for sample</li></ol>
</ol>
</ol>
Line 42: Line 42:
<ol type="A"><li>For VIM-PA and VIM-PB:</li>
<ol type="A"><li>For VIM-PA and VIM-PB:</li>
<ol type="a">
<ol type="a">
<li>Mix SMCC-treated antibodies with DTT-treated oligonucleotides at 10X molar excess of oligo to Ab</li>
<li>Mix SMCC-treated antibodies (22 uL) with DTT-treated oligonucleotides (35 uL)</li>
<ol type="i"><li>Amounts to 10X molar excess of oligo to Ab</li>
<li>Transfer to pre-wet [[Media:SlideALyzer_MINI_Dialy_Unit_UG_Manual.pdf|Slide-A-Lyzer Mini 7 MWCO dialysis cup]]</li>
<li>Transfer to pre-wet [[Media:SlideALyzer_MINI_Dialy_Unit_UG_Manual.pdf|Slide-A-Lyzer Mini 7 MWCO dialysis cup]]</li>
<li>Dialyze in 1 L PBS with 5 mM EDTA at 4C for 2 days with one buffer exchange to PBS</li>
<li>Dialyze in 1 L PBS with 5 mM EDTA at 4C for 2 days with one buffer exchange to PBS</li>
<li>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</li>
<li>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</li>
</ol>
</ol>

Latest revision as of 22:38, 26 May 2016

VIM Antibody Prep[edit]

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I found some columns Dinh had that were the product I've been waiting on, so I am going to use those to test out the antibody linkage and binding protocols. I'll use the VIM protein again, since it has the highest expression.

Protocol[edit]

  1. Prep
    1. Resuspend VIM-PA in 588 uL nfH2O
    2. Resuspend VIM-PB in 532 uL nfH2O
  2. Antibody Activation
    1. Resuspend VIM with 100 uL PBS (final conc 1 mg/mL)
    2. Do this twice:
      1. Equilibrate a 40 kDa spin column 4 times with 100 uL PBS, (100 mM pH 7.3)
        1. Spin at 1000xg for 2 min
      2. Add 20 uL antibody to the column and spin for 3 min at 1000xg; collect in a new tube
      3. Add 2 uL sulfo-SMCC to sample
      4. Incubate for 2 hr at 4C with 3 times intermittent mixing
      5. Equilibrate a new 40 kDa column with 100 mM phosphate buffer with 20 mM EDTA
      6. Transfer antibodies to new column and spin for 3 min at 1000xg
  3. Oligonucleotide Activation
    1. For VIM-PA and VIM-PB:
      1. Add 13 uL 100 uM oligonucleotide to tube
      2. Add 22 uL 40 mM DTT
      3. Incubate at 95C for 2 min followed by 1 hr at 37C
      4. Add 165 uL PBS with 20 mM EDTA
      5. Remove excess DTT using two consecutive Zeba 7 kDa columns equilibrated with 100 mM PBS (10X PBS)
        1. Spin at 1500xg for 1 minute for equilibrium; 2 min for sample
  4. Ab-Oligo Mixing
    1. For VIM-PA and VIM-PB:
      1. Mix SMCC-treated antibodies (22 uL) with DTT-treated oligonucleotides (35 uL)
        1. Amounts to 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