Mapping 250K/500K SNP assay

90 GeneChip ® Mapping 500K Assay Manual
QUANTITATE THE DILUTED PCR PRODUCT
To quantitate the diluted PCR product:
1. Measure the OD of each PCR product at 260, 280 and 320 nm.
OD280 and OD320 are used as process controls. Their use is
described under Process Control Metrics below.
2. Determine the OD260 measurement for the water blank.
3. Determine the concentration of each PCR product as follows:
A. Take 3 OD readings for every sample (1 from each optical plate;
P1, P2, P3 and P4 in Figure 4.5 on page 89).
OD1 = (sample OD) – (water blank OD)
OD2 = (sample OD) – (water blank OD)
OD3 = (sample OD) – (water blank OD)
B. Average the 3 readings for each sample to obtain an Average
Sample OD:
Average Sample OD = (OD1 + OD2 + OD3) ÷ 3
C. Calculate the undiluted sample concentration for each sample
using the Average Sample OD:
Sample concentration in µg/µL =
Average Sample OD ✕ (0.05 µg/µL) ✕ 100
Apply the convention that 1 absorbance unit at 260 nm equals
50 µg/mL (equivalent to 0.05 µg/µL) for double-stranded PCR
products. This convention assumes a path length of 1 cm. Consult
your spectrophotometer handbook for further information.
ASSESS THE OD READINGS
Follow the guidelines below for assessing and troubleshooting OD
readings.
Average Sample OD
A typical average sample OD is 0.5 to 0.7. This OD range is
equivalent to a final PCR product concentration of 2.5 to 3.5 µg/µL.
It is based on the use of a conventional UV spectrophotometer plate
reader and assumes a path length of 1 cm.