Low capture library amounts (less than 0.5 ng/ul) were obtained despite good amounts of individual libraries, why?
Ensure the following capture protocol recommendations are respected:
Ensure the use of:
– Low bind tubes
– Suitable 1.5 ml tube rotor
– Temperature settings between 45 and 50°C (we recommend 45°C with machine settings on medium).
The DNA pellet should be visible.
Wash buffer preparation
Ensure that all reagents used to prepare buffers are properly mixed (vortex). This is particularly important for “Wash Buffer I” that typically contains precipitates.
Hybridization and washes
Always use a calibrated thermocycler and verify heat block temperature with a calibrated thermometer.
– During the stringent washes follow the exact protocol timing for each step.
– Pipet gently.
– Never vortex.
Always use a completely thawed polymerase mix and ensure homogenization before use.
– Prepare small aliquots if numerous freeze/thaw cycles are required.
– Make sure to properly mix the PCR reaction on the beads.
DNA Clean up
AMPure beads must be equilibrated at room temperature.
– Do not over-dry the beads (no visible cracks) as this reduces the yield of recovered DNA.