Tomato (breaker-stage) vs. Pepper (breaker-stage)
Hybridization: Tomato Breaker/Pepper Breaker
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Hybridization protocol
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| Hybridization solution | Corning Universal Hybridization Solution (catalog #40090, Corning Inc.) |
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| Solution concentration | Unknown due to Corning patent |
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| Blocking agent | Unknown due to Corning patent |
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| Wash procedure | 1) Wash arrays in 1X SSC/0.2% SDS for 10 minutes at 43 C (in the dark).
2) Wash arrays in 0.1X SSC/0.2% SDS for 10 minutes at room temperature (in the dark, with shaking).
3) Wash arrays in 0.1X SSC for 10 minutes at room temperature (in the dark, with shaking). |
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| Labeled Target Quantity | All (see instructions in Invitrogen labeling kit) |
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| Hybridization Time | 12 - 16 hours |
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| Hybridization Volume | 65 ul |
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| Hybridization Temperature | 43C |
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| Hybridization Instrument | Corning microarray hybridization chamber in 43C incubator |
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Scan information
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| Scan hardware | Scan Array 5000 |
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| Scan software | Scan Array ver 3.1 |
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Image analysis information
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| Software | ImaGene ver 5.5 |
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| Analysis algorithm | 1) Images
Cy5 = Red Cy3 = Green
2) Spot Finding
Find negative spots = no
Enforce grid constraints = yes
Local flexibility = 6.0
Global flexibility = 75%
3) Segmentation
Background buffer = 2.25
Background width = 5.0
Autosegmentation = no
Signal low cutoff = 20%
Signal high cutoff = 100%
Background low cutoff = 0%
Backgound high cutoff = 88%
4) Quality Flags
Empty spots = no
Negative spots = yes
Poor spots = yes
5) Poor Spot Parameters
Contamination of background/signal = no
Ignored pixels percentage = yes (setting = 69%)
Open perimeter ratio = no
Shape regularity = no
Area to perimeter ratio = yes (setting = 0.6)
Offset value = yes (setting = 17.5) |
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Normalization information
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| Normalization software | marray package under R |
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| Normalization algorithm | Print-tip specific Lowess Normalization |
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| Normalization strategy | Total Array |
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