Solanaceae fruit comparison
Hybridization: Green Pepper/Ripe Pepper |
| Hybridization protocol |
| Hybridization solution | SDS Genisphere Hybridization Solution |
|---|
| Solution concentration | 0.25m NaPO4, 4.5%SDS, 1mm EDTA, 1x SSC, 2x Denhardt's Solution |
|---|
| Blocking agent | ssDNA and Oligo dT |
|---|
| Wash procedure | 2x15 minutes
1) 2x SSC + 0.2% SDS at 55 C.
2) 2x SSC at RT.
3) 0.2x SSC at RT (+ 0.5mM DTT) |
|---|
| Labeled Target Quantity | 6 ul labeled cDNA |
|---|
| Hybridization Time | (1) 14 hours (2) 2 hours |
|---|
| Hybridization Volume | 65 ul |
|---|
| Hybridization Temperature | 60 C / 55 C for secondary hyb |
|---|
| Hybridization Instrument | Corning hybridization chambers in water bath |
|---|
|
| Scan information |
| Scan hardware | Scan Array 5000 |
|---|
| scan software | Scan Array ver 3.1 |
|---|
|
| Image analysis information |
| Software | ImaGene ver 5.5.4 |
|---|
| Analysis algorithm | 1) Images
Cy5 = Red Cy3 = Green
2) Grid Flexibility
Enforce grid constraints = yes
Local flexibility = 6.0 pixels
Flexibility = 75%
3) Quality Control
Empty spots = no
Poor spots = yes
Find negative spots = no
4) Measurements
Background buffer = 2.25
Background width = 5.0
Auto seqmentation = no
Signal low cutoff = 20%
Signal high cutoff = 100%
Background low cutoff = 0%
Background high cutoff = 88%
5) Poor Spot Parameters
Contamination of Background/Signal = no
Ignored pixels % = yes (setting = 69)
Open perimeter = no
Shape regularity = no
Area to perimeter = yes (setting = 0.6)
Off-set value = yes (setting = 17.5) |
|---|
|
| Normalization information |
| Normalization software | marray package under R |
|---|
| Normalization algorithm | Print-tip specific Lowess Normalization |
|---|
| Normalization strategy | Total Array |
|---|
|
