Why does the CytoScan Assay User Guide state on page 35 to always prepare Fragmentation Master Mix according to Table 9 even when processing less than 24 samples?
It is critical to have correct size of the fragments for an optimal hybridization. The fragmentation reagent is very viscous, and it is easier to get a correct proportion of enzyme when a larger volume is used. Therefore, our recommendation is to always prepare a master mix for 24 samples, even if a smaller number of samples are processed.
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What are the recommended requirements for genomic DNA samples used in the CytoScan assay?
The tested sources of human gDNA used in the CytoScan Assay are blood and cell line samples.
The genomic DNA (gDNA) sample must be double-stranded, not degraded and free of any contaminants (e.g., PCR inhibitors and other human/non-human gDNA). The recommended starting amount is 250 ng (5 µL with 50 ng/µL) dissolved in low EDTA TE buffer. High EDTA concentration may negatively impact the downstream enzymatic reactions. We recommend running the gDNA samples on a 0.8-1% agarose gel for side-by side comparison with a control DNA (included in the kit). High-quality genomic DNA will run as a major band at approximately 10-20 kb on the gel.
For more detailed information please refer to the CytoScan Assay User Guide, page 9, Genomic DNA general requirements and recommendations section .
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Which type of freezer should be used to store CytoScan Reagent kit components that require -20 degrees C?
The reagents that require storage at -20 degrees C must not be stored in a frost-free freezer. The activity of the enzymes will be decreased.
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What is the maximum number of freeze/thaw cycles recommended for the CytoScan Reagent kit?
The CytoScan Reagent kit has been validated for less than or equal to 5 freeze/thaw cycles.
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Why does the protocol state to prepare a fragmentation master mix enough for 24 samples even if a smaller number of samples is processed?
It is critical to have correct size of the fragments for an optimal hybridization. The fragmentation reagent is very viscous and it is easier to get a correct proportion of enzyme when a larger volume is used. The recommendation is to always prepare a master mix for 24 samples even if a smaller number of samples are processed.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.