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216 GeneChip ® <strong>Mapping</strong> <strong>500K</strong> Assay Manual<br />
Problem Likely Cause Solution<br />
Fragmented PCR product is not the correct size<br />
PCR product is still visible<br />
in 200-1,100 bp size region<br />
.CEL file can not be generated<br />
GCOS is unable to align<br />
grid.<br />
Incomplete fragmentation due to<br />
underestimated DNA concentration.<br />
Failed or incomplete fragmentation<br />
due to reduced DNase activity.<br />
Unable to place a grid on the .dat file<br />
due to the absence of B2 signal.<br />
.dat image is dim. Insufficient signal intensity or<br />
staining failure.<br />
Low <strong>SNP</strong> call rates<br />
Gel images and<br />
spectrophotometric<br />
quantitation indicate<br />
successful PCR reaction.<br />
Extremely low call rate<br />
Sample hybridization is<br />
absent on .cel and .dat<br />
images but B2 grid is<br />
bright.<br />
Incorrect wash buffers used on<br />
fluidics station.<br />
Over fragmentation of DNA sample<br />
due to incorrect dilution of<br />
Fragmentation Reagent (DNase I)<br />
stock.<br />
Ensure spectrophotometer is properly<br />
calibrated so only 90 µg of DNA is added to<br />
the fragmentation reaction.<br />
Check that you have entered the correct<br />
activity of DNase into the formula for<br />
calculating amount of DNase to add to<br />
fragmentation reaction. (See Dilute the<br />
Fragmentation Reagent on page 102;<br />
amount is the same for high and low<br />
throughput.)<br />
Ensure fragmentation reagent (DNase I) is<br />
kept at –20°C. Do not reuse diluted working<br />
stock.<br />
Hybridization controls including oligo B2<br />
must be added to hybridization cocktail for<br />
grid alignment.<br />
Make fresh stain buffers.<br />
Prime the fluidics station with the correct<br />
buffers prior to running the <strong>assay</strong>.<br />
Incorrect wash buffers will disrupt<br />
hybridization of the labeled, fragmented<br />
DNA.<br />
Use correct concentration of<br />
Fragmentation Reagent (DNase I). Check<br />
U/µL on the label and check dilution<br />
formula (Dilute the Fragmentation<br />
Reagent on page 102; amount is the same<br />
for high and low throughput). Work quickly<br />
and on ice; transfer reaction tubes to preheated<br />
thermal cycler (37°C). Mix<br />
thoroughly.<br />
Labeling reaction suboptimal. Use a new vial of Terminal<br />
Dideoxynucleotidyl Transferase. Verify the<br />
labeling reagents and repeat labeling.
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