Copy Number and Cytogenetics Support—Troubleshooting

Several things can cause this problem. The first step in troubleshooting this problem is to determine if the positive control worked. Common reasons for this failure include incomplete digestion of genomic DNA or inefficient ligation of adaptors, ligation samples that are not properly diluted or mixed, and degraded DNA (if only the positive control worked). See the troubleshooting section of the CytoScan™ Assay User Manual (Cat. No. 901808) for more information.

A refrigerated centrifuge is highly recommended, but if one is not available, you may use a plastic plate rack that has been stored at -20°C. We recommend that you keep the samples chilled and work quickly prior to initiating the incubation at 37°C. See the CytoScan™ Assay and Data Analysis Training Video for more details about required equipment and consumables.

Common causes of over fragmentation include excess enzyme due to pipetting errors or an incorrect volume of enzyme used based on the unit activity. In addition, warming up of the assembled reaction prior to initiating the 37°C incubation can lead to over fragmentation. See the troubleshooting section of the CytoScan™ Assay User Manual (Cat. No. 901808) for more information about over fragmentation.

Common causes of under fragmentation include improper storage or handling of the enzyme, an incorrect volume of enzyme used based on the unit activity, and improper mixing of fragmentation master mix. See the troubleshooting section of the CytoScan™ Assay User Manual (Cat. No. 901808) for more information about under fragmentation.

MAPD failures can be caused by assay drift due to variation in assay execution or over fragmentation. See the troubleshooting section of the CytoScan™ Assay User Manual (Cat. No. 901808) for more information.

Waviness SD failures can be caused by degraded DNA, an incompatible sample type, or a sample- specific effect. See the CytoScan™ Assay User Manual and the Chromosome Analysis Suite User Manual for more information.

After any stage in the assay (including fragmentation), samples can be stored at –20°C if you are not proceeding directly to the next step. However, once a stage has been initiated, it must be completed before storage of the samples.

A plate can be held in thermal cycler at 4°C for up to 60 hours.

A plate can be frozen at –15 to –25°C for up to one week.

The digestion step could be root cause. If the reaction did not go to completion, there may be, on average, longer fragments at the end of the reaction. These longer fragments then pass on to ligation and PCR reactions. Insufficient amounts of PCR primer were added to facilitate suppression PCR. The primer concentration shifts the PCR reaction equilibrium toward larger fragment distributions, in this case, by increasingly unfolding (linearizing) the stem-loop structure at increasing primer concentration when the same adaptor ends hybridize.

A smear in the negative control indicates there has been a contamination. If the negative control band is a light smear, it could be a low-level environmental bacterial contaminant introduced through plastic surfaces. This has shown not to have a material impact on the CytoScan™ HD data. If a bright band or a smaller smear is seen, then that could be a real cross-contaminant from a sample, and the samples would need to be checked for contamination. If a smear is seen in the negative control well, the negative control should be re-run to check if it was not a result of sample bleed over from gel loading.

Yes, the purified PCR products can be stored at –15 to –25°C, and one can continue with the purification step later.

The purified PCR products can be stored at –15 to –25°C for 10 days.

Arrays must be put onto the fluidics station immediately after removal from the hybridization oven. Do not remove arrays from the oven you are ready to wash them.

After the washing and staining step, arrays can be stored for up to 24 hours at 4°C before scanning.