TABLE 2.

Troubleshooting table.

Step	Problem	Solution
Step 13: DOP-PCR amplification	No amplification products	Ensure that BSA and β-mercaptoethanol have been added to the reaction and the pH of the TAPS solution has been adjusted to 9.3
	Negative control contaminated	It is crucial to UV-sterilize reagents as well as plastic ware before use. To rule out possible contamination of primers, we order our primers freeze-dried and dilute them before use in UV-sterilized water
Step 17: amino-linking PCR	Amino-linking PCR: no amplification product	Ensure that the amino-linking buffer has been prepared before use. Storage below room temperature results in inefficient amplification
Step 17: amino-linking PCR	Amino-linking PCR: negative control contaminated	Order primer freeze-dried and dilute before use in UV-sterilized water. Ensure you are working with sterile reagents and plastic ware
Step 39: random primed labeling	Fragments too small after random primer labeling	Ensure that DNA is not degraded, if necessary repeat DNA isolation
Step 39: random primed labeling	Insufficient incorporation of Cy-dyes	This can be due to degraded DNA used as a template or low DNA quality. If necessary, clean up DNA by, for example, phenol–chloroform extraction or repeat DNA isolation
Step 40: hybridization	Compressed hybridization ratios/increased ratio variability	A critical factor for the reliable detection of copy number changes is the sufficient suppression of repeat sequences during hybridization. This is dependent on the quality of the Cot1 DNA, which mainly consists of LINE and SINE repeats. When using high-quality Cot1 DNA, single copy changes are reported by log 2 ratio values close to 0.58 (single copy gain) and −1 (single copy loss). Low-quality Cot1 DNA will fail to suppress repeats completely, thus resulting in compressed abnormal ratios and increased background ratio variability The conditions of the second wash step (42 °C in 50% formamide/2× SSC for the manual procedure, 54 °C in 0.1× SSC for the automated one) are critical for efficient removal of nonspecific probe hybridization. Carefully adjust these conditions in case of compressed abnormal ratios, increased background ratio variability or low spot intensities
Step 40: hybridization	Noisy hybridization profiles	There are many factors contributing to noisy hybridization profiles. However, DNA quality, array quality and hybridization conditions (e.g., high humidity levels throughout hybridization, inappropriate washing conditions) as well as environmental factors such as temperature, humidity and high ozone levels are the main causes. Although environmental factors seem to influence the signal intensities especially with regard to Cy5, problems with hybridization conditions often result in speckly images. The addition of antioxidant in hybridization and wash solutions (such as cysteamine; see automated procedure) can help protect Cy5 from degradation and limit experimental variability It should also be noted that commercially available scanners without dynamic focusing if poorly adjusted can introduce spatial artifacts into the data especially when slide surfaces are uneven. This effect however can be reduced by performing block normalization analyses