Mapping 250K/500K SNP assay

266 GeneChip ® Mapping 500K Assay Manual
STEP 6: Quantification of Purified PCR Product
The spectrophotometer should be calibrated regularly to ensure
correct readings.
Due to the high PCR yield, the accuracy of the O.D. measurement is
critical. Carefully follow the steps below and be sure the O.D.
measurement is within the linear range of the instrument.
Prepare at least three independent dilutions of each sample for
accurate concentration measurement. Average the results before
proceeding.
1. Use spectrophotometric analysis to determine the purified PCR
product yield. If available, a plate reader is preferred for efficient
DNA concentration determination.
2. Add 2 µL of the purified PCR product to 198 µL molecular biology
grade water (100-fold dilution) and MIX WELL.
3. Read the absorbance at 260 nm. Ensure that the reading is in the
quantitative range of the instrument (generally 0.2 to 0.8 OD).
4. Apply the convention* that 1 absorbance unit at 260 nm equals
50 µg/mL for double-stranded PCR product.
*This convention assumes a path length of 1 cm. Consult your spectrophotometer handbook for
further information.
5. For fragmentation:
A. Transfer 90 µg of each of the purified DNA samples to the
corresponding wells of a new plate.
B. Bring the total volume of each well up to 45 µL by adding the
appropriate volume of RB Buffer.
C. Cover the plate with PCR plate cover film and seal tightly.
D. Vortex at medium speed for 2 seconds, and spin down at
2000 rpm for 1 minute.