Question: How are the ratios of SPRIselect beads calculated in 10x assays?
Answer: The calculation of the ratio of SPRIselect beads : DNA (reaction volume) indicated in User Guide of CG000204 (3’ v3.1) is used as an example to explain how one double-sided size selection is performed in two steps as follows.
During the v3.1 fragmentation reaction, you will:
Therefore, your total volume of post fragmentation, end-repair, and A-tailing is 50 μl. Then in step 3.2, you will add 30 μl of SPRIselect beads to the 50 μl of reaction.
With the ratio of SPRIselect beads : reaction volume as 0.6X (30:50), DNA fragments larger than 600 bp preferentially bind to SPRIselect beads. After incubation, you will place the reaction tube strip on a magnetic stand to pellet the SPRIselect beads. You will then transfer 75 μl of the supernatant (containing DNA fragments smaller than 600 bp) into a fresh tube and add 10 μl of SPRIselect beads to make the ratio of SPRIselect : reaction volume as 0.8X, calculated as follows.
For note, the volume of supernatant (75 μl) is not used in the calculation. Instead, you will use the total volume of SPRIselect added (30 μl + 10 μl) over the original volume of the sample (50 μl) to achieve a final ratio of 0.8. With 0.8X, the fragments larger than 200 bp preferentially bind to SPRIselect beads. You keep the beads (with the DNA bound) and discard the supernatant, leading to the eluted fragments ranging from 200 to 600 bp.
In summary, as illustrated in the following schematic overview (Fig. 1), during 1st SPRI clean-up at 0.6X, SPRI beads selectively bind DNA fragments larger than 600 bp and the beads are discarded. During 2nd SPRI clean-up, the resulting supernatant (with DNA fragments smaller than 600 bp) is mixed with a fresh volume of SPRIselect beads to make a SPRIselect : reaction volume ratio of 0.8X. At 0.8X, SPRI beads selectively bind to the DNA fragments larger than 200 bp. At the 2nd SPRI clean-up, the beads are kept and the DNA is eluted. After 2 steps of SPRI clean-ups, the final sample with a tight fragment size distribution (200 – 600 bp) are achieved.