Mapping 250K/500K SNP assay

92 GeneChip ® Mapping 500K Assay Manual
Table 4.24
PROBLEM: Average Sample OD is Less Than 0.5 (2.5 µg/µL)
If the average sample OD of three independent measurements is less than 0.5 (calculated concentration less
than 2.5 µg/µL), a problem exists with either the genomic DNA, the PCR reaction, the elution of purified PCR
products, or the OD readings.
Possible problems with input genomic DNA that would lead to reduced yield include:
The presence of inhibitors (heme, EDTA, etc.).
Severely degraded genomic DNA.
Inaccurate concentration of genomic DNA.
NOTE: Check the OD reading for the PCR products derived from RefDNA 103 as a control for these
issues.
To prevent problems with the PCR reaction that would lead to reduced yield:
Use the recommended reagents and vendors (including AccuGENE® water) for all PCR mix
components.
Thoroughly mix all components before making the PCR Master Mix.
Pipette all reagents carefully, particularly the PCR Primer, when making the master mix.
Check all volume calculations for making the master mix.
Store all components and mixes on ice when working at the bench. Do not allow reagents to sit at
room temperature for extended periods of time.
Be sure to use the recommended PCR plates. Plates from other vendors may not fit correctly in the
thermal cycler block. Differences in plastic thickness and fit with the thermal cycler may lead to
variance in temperatures and ramp times.
Be sure to use the correct cycling mode when programming the thermal cycler (maximum mode on
the GeneAmp® PCR System 9700; calculated mode on the MJ Tetrad PTC-225).
Be sure to use silver or gold-plated silver blocks on the GeneAmp® PCR System 9700 (other blocks
are not capable of maximum mode, which will affect ramp times).
Use the recommended plate seal. Make sure the seal is tight and that no significant evaporation
occurs during the PCR.
NOTE: The Mapping 500K PCR reaction amplifies a size range of fragments that represents 15-20% of the
genome. The Mapping 500K arrays are designed to detect the SNPs that are amplified in this complex
fragment population. Subtle changes in the PCR conditions may not affect the PCR yield, but may shift the
amplified size range up or down very slightly. This can lead to reduced amplification of SNPs that are
assayed on the array set, subsequently leading to lower call rates.