Mapping 250K/500K SNP assay

86 GeneChip ® Mapping 500K Assay Manual
Table 4.22
Reagents Required for Stage 6: Quantitation and Normalization
Quantity Reagent
As needed RB Buffer (from Clontech DNA Amplification Clean-Up Kit)
75 mL AccuGENE® water, molecular biology-grade
IMPORTANT INFORMATION ABOUT THIS STAGE
To help ensure the best results, carefully read the information below
before you begin this stage of the protocol.
• Prepare three independent dilutions of each sample for accurate
concentration measurement. Average the results for each individual
sample before normalizing.
• The sample in each well must be normalized to 2 µg/µL in
RB Buffer (90 µg in 45 µL RB Buffer). Do NOT determine an
average concentration to use for every well.
• The amount of DNA added to the arrays has been optimized for the
best performance. Since not all wells will contain the same amount
of DNA after purification, the eluted PCR products must be
carefully normalized to 2 µg/µL before continuing to Stage 7:
Fragmentation.
• Normalize samples using RB Buffer (not water) to maintain the
correct pH for subsequent steps.
• The accuracy of the OD measurement is critical. Carefully follow the
steps below and be sure the OD measurement is within the
quantitative linear range of the instrument (0.2 to 0.8 OD).
• The spectrophotometer plate reader should be calibrated regularly
to ensure correct readings.
• This protocol has been optimized using a UV spectrophotometer
plate reader for quantitation.
NOTE: The NanoDrop® will give different quantitation results. This
protocol has not been optimized for use with this instrument. In