Mapping 250K/500K SNP assay

212 GeneChip ® Mapping 500K Assay Manual
4. Fragmentation Reagent (DNAse I) activity can decline over time
after dilution on ice, and so the reagent should be added to samples
as quickly as possible.
5. The use of master mixes prepared with an excess (15% for 96-well
plate protocol; 5% for low throughput protocol) ensures
consistency in reagent preparation by minimizing pipetting errors
and reducing handling time of temperature sensitive reagents. The
success of this assay depends on the accurate pipetting and
subsequent thorough mixing of small volumes of reagents.
6. The PCR reaction for this assay has been validated using one of the
specified thermal cyclers. These thermal cyclers were chosen
because of their ramping times. We highly recommend the PCR
thermal cyclers be calibrated regularly. Take care programming
your thermal cycler and use the thin walled reaction tubes
recommended. Thicker walled tubes may result in reduced PCR
efficiency and lower yields.
7. It is essential to run gels to monitor both the PCR reaction and the
fragmentation reaction.
For the PCR reaction, individual PCR products are run on a 2%
agarose gel – product (bands) should be visible in the 200 to 1,100
bp size range. See Figure 4.3 on page 73.
Following fragmentation, run samples on a 4% agarose gel.
Successful fragmentation is confirmed by the presence of a smear
of less than 200 bp in size, shown in Figure 4.7 on page 106.
8. Run controls in parallel with each group of samples.
Substitute water for DNA at the PCR step as a negative control.
The absence of bands on your PCR gel for this control confirms no
previously amplified PCR product has contaminated your samples.
The Reference Genomic DNA 103 is supplied as a positive control
in the assay kits. This is an effective troubleshooting tool
confirming all individual steps have been successful completed.
9. Oligonucleotide controls are included in the assay kit, these are
added to the target samples prior to hybridization and act to
confirm successful hybridization, washing, staining, and
sensitivity of the array. The oligonucleotide control reagents
contain oligo B2 which is used for grid alignment.