|
Purpose: These dyes can be used to stain the total DNA (all
spots) on test arrays in order to check that the DNAs have been
spotted. Alternatively, they can be used to stain a slide having
questionable results after hybridization in order to check that
the reson was not the absence of enough DNA spotted on the array.
Fluorescence:
| Dye |
Absorbance |
Emittance |
Corresonds to |
| PoPo-3 (pink) |
534 |
570 |
Channel 1 |
| Cy3 |
555 |
565 |
Channel 1 |
| ToTo-3 (blue) |
642 |
675 |
Channel 2 |
| Cy5 |
650 |
665 |
Channel 2 |
| ScanArray 3000 Lasers: |
Channel 1: |
543 nm |
| |
Channel 2: |
633 nm |
Be sure that your array that has been UV cross linked and
blocked
| 1. |
Dilute 1 mM dye 1:33 in DMSO to have 30 uM
working stock. |
| 2. |
Dilute 30 uM stock 1 in 30 in 1X PBS to have
1uM dye solution. |
| 3. |
Vortex. Quick spin. |
| 4. |
Add 1 uM dye to cover slip (HybiSlip) |
|
| |
22 x 22 |
15 ul |
| |
40 x 22 |
30 ul |
| |
60 x 22 |
45 ul |
| 5. |
Add arrayed slide to the cover slip. |
| 6. |
Place into chamber. Wrap with foil |
| 7. |
Incubate at RT in dark for 30 min. |
| 8. |
Wash array twice for 10 min in 1X PBS with
shaking or stirring |
| 9. |
Spin dry for 2 minute at 500 rpm |
Reagents:
PoPo-3 (P-3584, Molecular Probes)
ToTo-3 (T-3604, Molecular Probes)
(smaller quantities in: Nucleic Acid Stains Dimer Sampler Kit
N-7565)
PBS Maniatis recipe for 10X PBS
|
80g
|
NaCl |
|
2g
|
KCl |
|
14.4g
|
Na2HPO4 |
|
2.4g
|
KH2PO4 |
|
800 mls
|
H2O |
pH to 7.4 with 10M NaOH
Add H2O to 1000 mls
Filter sterilize (also removes lint)
|
