indexedlibrarypreparationandagilentcapturearrayhybridi.docVIP

Indexed library preparation and Agilent capture array hybridization for Illumina Genome Analyzer sequencing Scope and Purpose: To prepare genomic DNA libraries with flanking, indexed primers for pooling and target locus enrichment on an Agilent DNA capture array, followed by sequencing on an Illumina Genome Analyzer. Equipment and Supplies: (see Meyer and Kircher 2010, and Hodges et al. 2009 for reagent catalog numbers) Reagents and Equipment for DNA quantification DNA tube racks PCR plate racks Multichannel reagent basins Pipettes (10ul, 100ul, 1000ul, and multichannel (20ul, 200ul)) 10X Oligo hybridization buffer IS1_P5_short primer (500uM) IS2_P7_short primer (500uM) IS3_P5P7_comp primer (500uM) MilliQ water Water (HPLC grade) Tubes (strip, 0.6ml, 1.7ml) EB Buffer Bioruptor UCD-200 Crushed ice Reagents and equipment for agarose gel electrophoresis 10X Tango buffer ATP- 100 mM dNTP mix – 10 mM T4 polynucleotide kinase T4 DNA polymerase Thermocycler 10 or 50ml falcon tubes SPRI-XP Bead Mix Tween-20 EBT buffer 70% Ethanol Mini vortexer Mini centrifuge for strip tubes Centrifuge for 96-well plates Agencourt SPRI Plate Super Magnet Plate VorTemp 56 (Labnet #S2056A) 10X T4 DNA ligase buffer PEG-4000 (50%) T4 DNA ligase 10X Thermopol buffer Bst polymerase Sol_PCR_P5 primer Real-time PCR cycler-Mx3005P Qpcr machine Syber green I (1:3000 in TE and stock solution) Rox std (1:2500 in TE) 5x Phusion HF buffer Phusion Hot Start High-Fidelity DNA Polymerase Indexing primers (one for each LAB ID) Speed Vacuum Two heat blocks (for 1.7 tubes) Hybridization oven Two slide chambers for the hybridization oven Wash buffer 1 Wash buffer 2 Three slide staining dishes Slide rack Glass drying tray Plastic bottle (1 liter) BO1 primer (200uM) BO3 primer (200uM) BO3.P7.part1.F primer (200uM) BO4.P7.part1.R primer (200uM) BO5.P7.part2.F primer (200uM) BO6.P7.part2.R primer (200uM) COT-1 DNA 10X Agilent blocking agent 2X Agilent hybridization buffer Two gasket slides O