Xpert DNA Purification Magnetic Beads

Protocols

1. Add Xpert Magnetic Beads to the DNA samples according to the tables below. For other volumes, simply modify proportionally using a beads-to-DNA ratio of 1.8

96-well microplate 384-well microplate
sample (µl) beads (µl) sample (µl) beads (µl)
5 9 2 3.6
10 18 5 9
20 36 7 12.6
50 90 10 18
100 180 12.5 22.5

2. Mix thoroughly to a homogenous appearance by pipetting up and down the entire mixture 10 times. Allow for optimal DNA binding by incubation at room temperature for 5 minutes.
3. In order to separate the magnetic beads from the solution, place the microplate onto a magnetic separation rack for 5 minutes (Ensure the solution has becomes clear before proceeding to step 4).

During steps 4 to 8 maintain the microplate on the magnetic separation rack at all times.
4. Carefully aspirate off the cleared solution and discard. Avoid disturbing the magnetic beads.
5. Dispense 70% ethanol to each well: 200µl/well in case of a 96-well plate or 30µl/well in case of a 384-well plate.
6. Incubate at room temperature for 30 seconds.
7. Carefully aspirate off the ethanol and discard. Avoid disturbing the magnetic beads.
8. Repeat steps 5-7 once, then allow the plate to air-dry for 3-5 minutes to remove any residual ethanol.

9. Remove the microplate from the magnetic rack and elute DNA with 10-50µl/well (as desired) of elution buffer (e.g., nuclease-free water, TE, 10mM Tris-HCl pH 8.0 or 10mM Tris-acetate pH 8.0).
10. Mix thoroughly by pipetting up and down the entire mixture 10 times.
11. Incubate at room temperature for 2-5 minutes.
12. Place the microplate back onto the magnetic separation rack for 5 minutes.
13. Carefully transfer the eluent to a new plate. In case of carryover of beads, repeat step 12 and transfer into another new plate (or tube).
14. Store purified DNA at -20ºC or proceed with downstream application.

Size Selection

The basic protocol is optimized for PCR Clean-up. However, the protocol can be easily adapted for other applications by using different beads:DNA volume ratios. In general, the higher the ratio the smaller the fragments that are captured. For example, using a ratio of 0.8x instead of 1.8x would results in the removal of DNA fragments smaller than 250bp (instead of smaller than 100bp), or alternatively the removal of DNA fragments that are larger than 250bp if the unbound fraction was retained.

Table 1. Recommended Beads-to-DNA ratio for Size Selection
Component 150bp 200bp 250bp 300bp 400bp 500bp 600bp 700bp
Beads-to-DNA ratio 1.50 0.90 0.80 0.70 0.60 0.55 0.50 0.45

In the table above, one can find the recommended beads-to-DNA ratio for the purification of DNA above a desired minimum size. For example, when starting with a 50µl sample and using 45µl of Xpert DNA Purification Magnetic Beads (ratio of 0.90), the final product would be purified DNA of size of 200bp and larger, whereas if one would use 40µl of beads (ratio of 0.80), the final product would not include 200bp sized DNA but only DNA of 250bp and larger. And so on. So, one just needs to adapt step 1 of the basic protocol to the desired beads-to-DNA ratio. It is highly recommended to use samples of at least 50µl, as lower volumes will decrease pipetting accuracy and hence increase selection point variability.