NimbleGen SeqCap EZ Library SR User's Guide - Roche Applied ...

Table of ContentsPreface .................................................................................................................................... 5Intended Use ................................................................................................................................... 5SeqCap EZ Library .......................................................................................................................................... 5Contact Information ...................................................................................................................... 5Technical Support ........................................................................................................................................... 5Manufacturer and Distribution ..................................................................................................................... 5Conventions Used in This Manual .............................................................................................. 6Symbols ............................................................................................................................................................. 6Text ................................................................................................................................................................. 6Chapter 1. Before You Begin .............................................................................................. 7Workflow ......................................................................................................................................... 7What’s New? .................................................................................................................................... 8Terminology .................................................................................................................................... 8Components Supplied .................................................................................................................... 9Protocol Information & Safety ...................................................................................................... 9Storage of the SeqCap EZ Library ................................................................................................ 9Required Equipment, Labware & Consumables ........................................................................ 9Laboratory Equipment ................................................................................................................................... 9Consumables Available from Roche Applied Science ............................................................................. 10Consumables Purchased from Other Vendors ......................................................................................... 10Custom Oligonucleotides Purchased from IDT or Another Vendor ................................................... 10Chapter 2. Storing the SeqCap EZ Library ..................................................................... 13Chapter 3. Preparing the Sample Library and Performing QA .................................. 15References ...................................................................................................................................... 15Step 1. Preparing the Sample Library ......................................................................................... 15Step 2. Performing Sample Library Quality Assessment ......................................................... 16Chapter 4. Amplifying the Sample Library Using LM-PCR ......................................... 17References ...................................................................................................................................... 17Sample Requirements ................................................................................................................... 17Step 1. Preparing the LM-PCR ................................................................................................... 17Step 2. Performing PCR Amplification ..................................................................................... 18Step 3. Cleaning up the Amplified Sample Library .................................................................. 18Step 4. Checking the Quality of the Amplified Sample Library ............................................. 19Chapter 5. Hybridizing the Sample and SeqCap EZ Libraries ................................... 21Step 1. Preparing for Hybridization ........................................................................................... 21Step 2. Prepare the Hybridization Sample ................................................................................ 21Chapter 6. Washing and Recovering Captured DNA ................................................... 23Step 1. Preparing Sequence Capture and Bead Wash Buffers ................................................ 23Step 2. Preparing the Streptavidin Dynabeads ......................................................................... 23Step 3. Binding DNA to the Streptavidin Dynabeads .............................................................. 24Step 4. Washing the Streptavidin Dynabeads Plus Bound DNA ........................................... 24Chapter 7. Amplifying Captured DNA Using LM-PCR ................................................. 27References ...................................................................................................................................... 27Step 1. Preparing the LM-PCR ................................................................................................... 27NimbleGen SeqCap EZ Library SR User’s Guide v3.03