title: Parachondrostoma toxostoma microsatellite library
design: An optimization of classical biotin-enrichment methods (Kijas et al. 1994) was used to prepare the enriched libraries. Genomic DNA was extracted from muscle, with the DNeasy Tissue Kit (QIAGEN).   Enrichment was carried out at Genoscreen (Lille, France), according to the following procedure. Genomic DNA (1 lg) was digested with RsaI (FERMENTAS) for 1 h at 37 _C, according to the manufacturer’s recommendations, and was then ligated to standard adapters (Adap-F: GTTTAAGGCCTAGCTAGCAGAATC and Adap-R: GATTCTGCTAGCTAGGCCTT).  Samples were then purified on a Nucleofast PCR plate (MACHEREY-NAGEL). Eight biotin-labelled oligonucleotides ((AG)10, (AC)10, (AAC)8, (AGG)8, (ACG)8, (AAG)8, (ACAT)6 and (ATCT)6), corresponding to eight targeted microsatellite motifs, were hybridized to the ligated DNA at 56 C for 20 min, after initial denaturation of the ligated DNA.   The enrichment step was completed with Dynabeads (INVITROGEN). The resulting enriched DNA was amplified with primers corresponding to the library adapters, over 25 cycles (20 s at 95 C, 20 s at 60 C and 90 s at 72 C) and a final extension step of 30 min at 72 C. The PCR products were purified with a QIAquick PCR purification kit (QIAGEN).
identifiers.namespace: Aix-Marseille University
identifiers.id: P_toxostoma_AIP6_GH