Mapping 250K/500K SNP assay

ABOUT THIS PROTOCOL
96-Well Plate Protocol
The 96-well plate protocol described in this chapter is designed for
processing one full 96-well reaction plate, one enzyme at a time. The
protocol is presented in stages:
• Stage 1: Genomic DNA Plate Preparation
• Stage 2: Restriction Enzyme Digestion
• Stage 3: Ligation
• Stage 4: PCR
• Stage 5: PCR Product Purification and Elution
• Stage 6: Quantitation and Normalization
• Stage 7: Fragmentation
• Stage 8: Labeling
• Stage 9: Target Hybridization
The protocol and guidelines include key points regarding the various
molecular biology steps that comprise whole-genome sampling
analysis (WGSA).
Successful performance of the various molecular biology steps in this
protocol requires accuracy and attention to detail. Many of these stages
involve specific yet distinct enzymatic reactions. For example, in stage
2 the genomic DNA is initially digested with the restriction enzymes
Nsp I or Sty I. In stage 3, it is ligated to a common adaptor with T4
DNA ligase. Following ligation, the template undergoes PCR (stage
4) using TITANIUM Taq DNA polymerase. Once the product has
been purified and normalized (stage 5), it is then fragmented in stage
7 with Fragmentation Reagent (DNAse I), and end-labeled using
terminal deoxynucleotidyl transferase (stage 8).
The stages involving enzymatic reactions are the most critical of the
assay. Thus, it is important to carefully monitor and control any
variables such as pH, salt concentrations, time, and temperature, all of
which can adversely modulate enzyme activity.
Successful sample processing can be achieved by incorporating the
following principles:
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