Time course of DC3000 vs delta hrp on tomato Prf-3
Hybridization: DC3000/delat hrp 0h |
| Hybridization protocol |
| Hybridization solution | Genisphere Hybridization Solution |
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| Solution concentration | 0.25m NaPO4, 4.5%SDS, 1mm EDTA, 1x SSC, 2x Denhardt's Solution |
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| Blocking agent | ssDNA and Oligo dT |
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| Wash procedure | 3x10 minutes:
1) 2x SSC + 0.2% SDS at 55C.
2) 2x SSC at 22 C.
3) 0.2x SSC at 22 C |
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| Labeled Target Quantity | 6ul |
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| Hybridization Time | 12-16 hours |
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| Hybridization Volume | 65ul |
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| Hybridization Temperature | 60 C |
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| Hybridization Instrument | Corning microarray hybridization chamber in water bath |
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|
| Scan information |
| Scan hardware | Scan Array 5000 |
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| scan software | Scan Array ver 3.1 |
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|
| Image analysis information |
| 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 |
| 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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