Mapping 250K/500K SNP assay

IMPORTANT INFORMATION ABOUT THIS STAGE
chapter 4 | 96-Well Plate Protocol 99
To help ensure the best results, carefully read the information below
before you begin this stage of the protocol.
• Purified PCR product must be normalized to 90 µg DNA in 45 µL
RB Buffer.
• The degree of fragmentation is critical. Perform this stage carefully
to ensure uniform, reproducible fragmentation.
• The Fragmentation Reagent is extremely temperature sensitive. It
rapidly loses activity at higher temperatures. To avoid loss of
activity:
- Dilute the Fragmentation Reagent immediately prior to use.
- Keep at –20 °C until ready to use. Transport and hold in a –20 °C
cooler. Return to the cooler immediately after use.
- Perform these steps rapidly and without interruption.
• The Fragmentation Reagent (DNase I) may adhere to the walls of
some microfuge tubes and 96-well plates. To ensure the accurate
amount of DNase I in the fragmentation reaction (Stage 7:
Fragmentation), the strip tubes used for this stage must be cut from
Bio-Rad 96-well unskirted PCR plates, P/N MLP-9601. See Cutting
Strip Tubes From Plates on page 41 and Table 4.1 on page 40 for more
information on these plates.
• The Fragmentation Reagent is viscous and requires extra care when
pipetting. Follow these guidelines:
- When aspirating, allow enough time for the correct volume of
solution to enter the pipette tip.
- Avoid excess solution on the outside of the pipette tip.
• Be sure to use the AccuGENE® water listed in Appendix A. Using
in-house ddH2O or other water can negatively affect your results.
The reaction in Stage 7: Fragmentation is particularly sensitive to pH
and metal ion contamination.
• All additions, dilutions and mixing must be performed on ice.