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=BSA Ab-Oligo Linkage-Version 3 Probes (Started [[Daniel:Notebook/ComboLock/2017-5-13|Saturday May 13]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] Repeat of Saturday's experiment for the "B" probe, which mysteriously vanished during one of the filtration runs. [[Daniel:Protocols/AmiconUltra|Original Amicon protocol]] ==Protocol== <ol> <li>Antibody Activation</li> <ol type="A"> <li>Resuspend antibodies at 1 mg/mL in PBS by adding 10 uL antibody to 10 uL PBS</li> <li>Equilibrate a 10K Amicon tube by adding 500 uL 100 mM phosphate buffer; centrifuge at 14000xg for 10 minutes</li> <li>Add 20 uL antibody and 450 uL 100 mM phosphate buffer to the column and spin for 15 min at 14000xg</li> <li>Extract the 22 uL using a pipette and add it to low protein bind 1.5 mL tube</li> <li>Dissolve sulfo-SMCC to 3.33 mM in 100mM phosphate buffer</li> <li>Add 2 uL sulfo-SMCC to sample and incubate for 2 hr at 4C with 3 times intermittent mixing</li> <li>Equilibrate a new 10 kDa Amicon column with 100 mM phosphate buffer with 20 mM EDTA; Centrifuge 14000xg for 10 minutes</li> <li>Add sample and 440 uL 100 mM PB with 20 mM EDTA; centrifuge at 14000xg for 15 minutes</li></ol> <li>Oligonucleotide Activation</li> <ol type="A"> <li>Resuspend oligonucleotide in 1 mM in 100 mM phosphate buffer with 20 mM EDTA</li> <li>Add 1.3 uL oligonucleotide to tube (or plate)</li> <li>Add 2.2 uL 40 mM DTT; incubate at 95C for 2 min followed by 1 hr at 37C</li> <li>Add 20 uL PBS with 20 mM EDTA</li> <li>Equilibrate a 10K Amicon tube by adding 475 uL PBS; centrifuge at 14000xg for 10 minutes</li> <li>Add sample and 440 uL PBS (5% DTT); centrifuge at 14000xg for 10 minutes</li> <li>Add 465 uL PBS (0.35% DTT); centrifuge at 14000xg for 10 minutes</li> <li>Add 465 uL PBS (0.02% DTT); centrifuge at 14000xg for 15 minutes</li></ol> <li>Ab-Oligo Mixing</li> <ol type="A"> <li>Mix SMCC-treated antibodies with DTT-treated oligonucleotides at 10X molar excess of oligo to Ab; wait 10 minutes</li> <li>Transfer to amicon Ultra centrifugal filter; add 450 uL PBS+ 5mM EDTA; Centrifuge 10 minutes at 14000xg</li> <li>Add 465 uL PBS; Centrifuge 10 minutes at 14000xg</li> <li>Add 465 uL PBS; Centrifuge 15 minutes at 14000xg</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>Store at 4C</li> </ol> </ol> [[Category:ComboLock]] [[Category:20170513]]
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