Mapping 250K/500K SNP assay

Assay Recommendations
211
Genotyping applications require very high accuracy to achieve
maximum power. Therefore, great care should be taken to avoid
possible sources of cross contamination that would lead to genotyping
errors. As with any assay using PCR, the GeneChip ® Mapping Assay
has an inherent risk of contamination with PCR product from previous
reactions. In Chapter 2, we recommend a workflow to minimize the
risk of cross contamination during the assay procedure. It is essential
to adhere to workflow recommendations. PCR reactions should only
be carried out in the main laboratory and personnel should not re-enter
the Pre-PCR Clean and PCR staging areas following potential
exposure to PCR product without first showering and changing into
clean clothes.
It is essential to carefully read and follow the protocol as written. This
assay has been validated using the reagents and suppliers listed,
substitution of reagents and shortcuts are not recommended as they
could result in suboptimal results. For example, always use
AccuGENE® water from Cambrex, and ligase and restriction enzymes
from New England Biolabs.
Additional recommendations are listed below:
1. Think ahead to ensure that reagents and equipment you require,
including designated pipettes, are in the correct work location.
This will make workflow easier and prevent contamination risks.
2. Check that your spectrophotometer is accurately calibrated and
ensure readings are in dynamic range (~ 0.2 to 0.8 OD). This will
ensure you use the correct amount of genomic DNA, necessary for
fragmentation reaction efficiency and identifying good PCR
yields. Adding the same amount of labeled target to the arrays will
help obtain reproducible call rates.
3. Pay particular attention to the storage and handling of reagents.
This is especially important for enzymes such as DNA Ligase and
GeneChip ® Fragmentation Reagent (DNase I) which are sensitive
to temperatures exceeding –20°C. To prevent loss of enzyme
activity when the enzymes are removed from the freezer use a
bench top freeze block and return to –20°C directly after use. Take
care when pipetting enzymes stored in glycerol, which is viscous.
Do not store at –80°C.