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=BSA Ab-Oligo Linkage-Version 3 Probes= [[Daniel:Notebook/ComboLock|Back to Calendar]] This experiment will be preparing the BSA Ab-Oligo linkage, which serves as the basis for the Antibody version of oligo capture. Note that you have to do 2 samples, one for the "A" oligo and one for the "B" oligo. The "A" oligo is the 5' thiol modified (equivalent to the C1 or PCCA probe) and the "B" oligo is 3' thiol modified, equivalent to the C2 or "B" probe. Another change is for dialysis and buffer exchange (the last few steps), I'll be switching from passive dialysis to using the [https://www.emdmillipore.com/US/en/product/Amicon-Ultra-0.5%C2%A0mL-Centrifugal-Filters-for-DNA-and-Protein-Purification-and-Concentration,MM_NF-C82301?ReferrerURL=https%3A%2F%2Fwww.google.com%2F Amicon Ultra 0.5mL Centrifugal filters], which provide a molecular weight cutoff filter to pass through dialysate while not allowing the larger molecules (in this case 10kDa which is pretty much every protein/DNA). See image below for general principal. [[Image:CentrifugalFilters.png|500px]] ==Protocol== <ol> <li>Buffer Prep</li> <ol type="A"><li>Dilute 1 M phosphate buffer by 10X</li></ol> <li>Antibody Activation</li> <ol type="A"> <li>Resuspend antibodies at 1 mg/mL in 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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