Proceed to perform the reaction after functionalizing both ends of the linear DNA substrate. First, take four S-400 spin columns and vortex them for at least 30 seconds to resuspend the resin. Then, centrifuge them for one minute at 735G to remove the storage buffer.
Transfer the columns to clean 1.5 milliliter tubes. Immediately, add 100 microliters of the doubly functionalized linear DNA solution to each column. Centrifuge the columns for two minutes at 735G to flow the DNA through the column while retaining the unincorporated nucleotides.
After pooling together the flow-through containing the DNA from the four columns, measure the volume with a pipette. Next, vortex M-280 streptavidin-coated magnetic beads for 30 seconds to resuspend them. Transfer four milligrams of the resuspended magnetic beads to a clean 1.5 milliliter tube.
Place the tube in a magnetic rack for one minute to collect the beads before removing the storage buffer. Resuspend the beads in one milliliter of buffer A by vortexing for five seconds before incubating the beads at room temperature for five minutes. Collect the beads by placing the tube in a magnetic holder for one minute.
After removing buffer A, resuspend the beads in 400 microliters of 2X buffer A by pipetting. Then, add 400 microliters of the functionalized DNA solution to the beads, and mix gently by pipetting. Incubate the bead DNA mixture overnight at four degrees Celsius with end over end rotation.
The next day, use the magnetic rack to remove the supernatant before calculating the total amount of DNA bound to the magnetic beads. Then, wash the beads sequentially with buffer B and buffer C.Resuspend the beads in 300 microliters of buffer C and make four 75 microliter aliquots.
