A-Tailing

If you are stopping here for a safe stopping point, resuspend the beads in 7 ul of 1x A Tailing buffer, otherwise, resuspend them in 15uL A-tailing master mix.

• Resuspend the beads in 15 ul 1X A-Tailing Buffer (see table above), cover the reaction and store at 4 oC for up to 24 hours. Do not freeze the samples as this will damage the AMPure® XP® beads. When ready, proceed to –A-Tailing after Safe Stopping Point–.

---

A-Tailing

• A-Tailing is performed either directly after the 2nd Strand Synthesis and Marking Cleanup, or after the Safe Stopping Point, where beads were resuspended in 1X A-Tailing Buffer and stored at 4 °C for up to 24 hours.

A-Tailing immediately

• Assemble the A-Tailing reaction as follows:

Component	Volume
Beads with dscDNA	–
A-Tailing Master Mix	15 ul
–Total Volume–	–15 ul–

• Mix thoroughly by pipetting up and down several times.
• Incubate the plate/tube using the following protocol:

Step	Temp.	Duration
A-Tailing	30 °C	30 min
Enzyme inactivation	60 °C	30 min
HOLD	4 °C	∞

• Proceed immediately to –Adapter Ligation–.

A-Tailing after safe stopping point

• To resume library preparation, combine the following reagents to perform A-Tailing:

Component	Volume
Beads with dscDNA (in 1X A-Tailing Buffer)	7.5 ul
A-Tailing Master Mix after Safe Stopping Point	7.5 ul
–Total Volume–	–15 ul–

• Mix thoroughly by pipetting up and down several times.
• Incubate the plate/tube using the following protocol:

Step	Temp.	Duration
A-Tailing	30 °C	30 min
Enzyme inactivation	60 °C	30 min
HOLD	4 °C	∞

• Proceed immediately to –Adapter Ligation–.