MboI R6711

Protocol tips

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	Genomic DNA was extracted from the pterygiophore which preserved by 70% ethanol, and then digested by restriction enzyme MboI (Promega) for the fragments were distributed at the 400– 900 bp.

Protocol tips
Genomic DNA was extracted from the pterygiophore which preserved by 70% ethanol, and then digested by restriction enzyme MboI (Promega) for the fragments were distributed at the 400– 900 bp.

Publication protocol

"Microsatellite markers were isolated followed the method of Quan et al. (2006). Genomic DNA was extracted from the pterygiophore which preserved by 70% ethanol, and then digested by restriction enzyme MboI (Promega) for the fragments were distributed at the 400– 900 bp. The partial genomic library constructed by the 400–900 bp DNA fragments was isolated by sucrose-density-gradient centrifugation, and then ligated to doublestranded adaptors sequences 50 -GATCGTCGACGGTACC GAATTCT-30 /50 -GTCAAGAATTCGGTACCGTCGAC-30 . After removed the superfluous adaptors, the partial genomic library was used as the templates for the first round of polymerase chain reaction (PCR). The PCR products were centrifugated to remove superfluous templates by centrifugal devices (Chang et al. 2005). The PCR products were primary hybridized to biotinylated probe of (CA)12, (GA)12
and (ACA)8 and the DNA hybridized to the probe were
separated and captured by streptavidin magnetic beads
(Dynal Biotech ASA, Norway). Then the enriched fractions
were used as the templates for the second round of PCR,
and the products were cloned using Easy TA Cloning Kit
(Promega). The plasmid library was transformed into
competent Escherichia coli DH5a utilizing the CaCl2
method (Sambrook and Russell 2001). The replicative
recombinant colonies onto nitrocellulose membranes
(Promega) were second hybridized by oligonucleotide 32Plabelled probes of (CA)12, (GA)12 and (ACA)8 to screen
positive clones."

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