FastDigest MluI

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Genomic DNA (1 μg) was digested for 20 min at 37°C with 1 μl of FastDigest MluI (Thermo Fisher Scientific), 1 μl of FastDigest SbfI (Thermo Fisher Scientific), and 2 μl 10× FastDigest buffer (Thermo Fisher Scientific) in a total volume of 20 μl.	The digestion was purified using AMPure XP beads (Beckman Coulter), with a ratio of 1.8:1 to the sample, followed by resuspension in 50 μl of ddH2O. Then, restriction‐associated double‐stranded Y adapters were ligated to the purified fragments in a 60 μl reaction containing 10 μM MluI adapter, 10 μM SbfI adapter, 5U T4 DNA ligase (Fermentas Inc.), 100 mM ATP, and 1× T4 ligation buffer.

Protocol tips
Genomic DNA (1 μg) was digested for 20 min at 37°C with 1 μl of FastDigest MluI (Thermo Fisher Scientific), 1 μl of FastDigest SbfI (Thermo Fisher Scientific), and 2 μl 10× FastDigest buffer (Thermo Fisher Scientific) in a total volume of 20 μl.
Downstream tips
The digestion was purified using AMPure XP beads (Beckman Coulter), with a ratio of 1.8:1 to the sample, followed by resuspension in 50 μl of ddH2O. Then, restriction‐associated double‐stranded Y adapters were ligated to the purified fragments in a 60 μl reaction containing 10 μM MluI adapter, 10 μM SbfI adapter, 5U T4 DNA ligase (Fermentas Inc.), 100 mM ATP, and 1× T4 ligation buffer.

Publication protocol

"The workflow of producing AFLP baits is illustrated in Figure ​Figure1.1. Because the quality of the genomic DNA is crucial for the outcome of AFLP experiment, we extracted high molecular weight DNA (fragments ~20 KB) from ethanol‐preserved tissue of an O. huanggangensis individual (Fujian) for AFLP bait preparation. Genomic DNA (1 μg) was digested for 20 min at 37°C with 1 μl of FastDigest MluI (Thermo Fisher Scientific), 1 μl of FastDigest SbfI (Thermo Fisher Scientific), and 2 μl 10× FastDigest buffer (Thermo Fisher Scientific) in a total volume of 20 μl. The digestion was purified using AMPure XP beads (Beckman Coulter), with a ratio of 1.8:1 to the sample, followed by resuspension in 50 μl of ddH2O. Then, restriction‐associated double‐stranded Y adapters were ligated to the purified fragments in a 60 μl reaction containing 10 μM MluI adapter, 10 μM SbfI adapter, 5U T4 DNA ligase (Fermentas Inc.), 100 mM ATP, and 1× T4 ligation buffer. Ligation reactions were incubated for 30 min at 25°C. The 60 μl ligation reaction was subsequently size‐selected for 500‐ to 2,000‐bp fragments with AMPure XP beads by separately adding 15 μl of beads (discard beads) and 20 μl of beads (keep beads) to the solutions. Finally, the size‐selected products were eluted from the air‐dry beads with 50 μl of 1× TE and used as templates in the next step.In the preamplification step, the design of Y adapters ensures that only those fragments ligated with both SbfI and MluI adapters at the two ends can be successfully amplified (Figure ​(Figure1).1). The PCR reaction mixture consisted of 1.25 U HiFi Taq DNA Polymerase (TransGen Inc.), 1× HiFi PCR buffer, 0.2 mM dNTPs, 0.2 μM MluI‐F primer, 0.2 μM SbfI‐R primer, and 5 ng template DNA in a total volume of 25 μl. The thermal cycling program included the following steps: an initial denaturation for 4 min at 94°C followed by 20 cycles of 30 s at 94°C, 1 min at 56°C, 1 min at 72°C, and a final extension of 10 min at 72°C. After this preamplification step, the reaction mixtures were diluted 10‐fold with 10 mM Tris‐HCl, 0.1 mM EDTA pH 8.0, and used as templates for the selective amplification.

Two forward (MluI‐F‐SA and MluI‐F‐ST) and two reverse (SbfI‐R‐SC and SbfI‐R‐SG) selective primers (each has a selective nucleotide at the 3′ end) were used in the selective amplification. So there were four primer combinations. To obtain single‐stranded baits in the next step, only the reverse selective primers were labeled with 5′ biotin. The reaction mixture for selective amplification contained 1 x HiFi PCR buffer, 0.2 mM dNTPs, 1.25 U HiFi Taq DNA Polymerase, 0.2 μM of forward selective primer, 0.2 μM reverse selective primer, and 2 μl of 10‐fold diluted preamplification product in a total volume of 25 μl. The thermal cycling program included the following steps: an initial denaturation for 4 min at 94°C followed by 36 cycles of 30 s at 94°C, 30‐s annealing, 1 min at 72°C, and a final extension of 10 min at 72°C. The annealing temperature in the first cycle was 65°C, then subsequently reduced in each cycle by 0.7°C for the next 12 cycles, and set at 56°C for the remaining 23 cycles. The selective amplification products were purified by AMpure XP beads and checked on a 1.2% TAE agarose gel. After that, four different selective amplification products (AFLP fragments) were pooled together in equal concentrations based on quantification with a NanoDrop 2000 spectrophotometer.

In the step of bait immobilization, 40 μl of biotinylated AFLP fragments (2 μg) was mixed with 40 μl of 2× BWT buffer (2 M NaCl, 10 mM Tris‐Cl, 1 mM EDTA, 0.1% Tween 20). The double‐stranded AFLP fragments were denatured by 5‐min incubation at 95°C and quickly transferred to ice bath. 50 μl of Dynabeads MyOne streptavidin magnetic beads (Life Technologies) was washed with 800 μl 1× BWT buffer and washed again with 800 μl TET buffer (10 mM Tris‐Cl, 1 mM EDTA, 0.05% Tween 20). The denatured AFLP fragments were then mixed with the washed MyOne beads and incubated for 20 min at 25°C to allow the biotinylated AFLP baits to bind to the beads. The beads were then washed four times with PWB buffer (0.1 M NaCl, 5 mM Tris‐Cl, 0.5 mM EDTA, 0.05% Tween 20) at 65°C to remove the nonbiotinylated strands. Finally, the bait‐coated beads were resuspended in 50 μl TET buffer and stored at 4°C."

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