| 1/2 | id: | 4 | | procedure_name: | Affy- Eukaryotic Array Washing and Staining | | instrument: | Fluidics Station 400 | | software: | MAS5.0 | | description: | CHAPTER 4 Eukaryotic Arrays: Washing, Staining, and Scanning 2.4.5
Eukaryotic
SECTION 2 Eukaryotic Sample and Array Processing 2.4.6
Reagent Preparation
Wash A: Non-Stringent Wash Buffer
(6X SSPE, 0.01% Tween 20)
For 1,000 mL:
300 mL of 20X SSPE
1.0 mL of 10% Tween-20
699 mL of water
Filter through a 0.2 µm filter
Wash B: Stringent Wash Buffer
(100 mM MES, 0.1M [Na+], 0.01% Tween 20)
For 1,000 mL:
83.3 mL of 12X MES Stock Buffer (see Section 2, Chapter 3 for reagent preparation)
5.2 mL of 5M NaCl
1.0 mL of 10% Tween 20
910.5 mL of water
Filter through a 0.2 µm filter
Store at 2°C to 8°C and shield from light
2X Stain Buffer
(Final 1X concentration: 100 mM MES, 1M [Na+], 0.05% Tween 20)
For 250 mL:
41.7 mL 12X MES Stock Buffer (see Section 2, Chapter 3 for reagent preparation)
92.5 mL 5 M NaCl
2.5 mL 10% Tween 20
113.3 mL water
Filter through a 0.2 µm filter
Store at 2°C to 8°C and shield from light
10 mg/mL Goat IgG Stock
Resuspend 50 mg in 5 mL 150 mM NaCl
Store at 4°C
If a larger volume of the 10 mg/mL IgG stock is prepared, aliquot and store at -20°C
until use. After the solution has been thawed it should be stored at 4°C. Avoid
additional freezing and thawing.
CHAPTER 4 Eukaryotic Arrays: Washing, Staining, and Scanning 2.4.7
Eukaryotic
Experiment and Fluidics Station Setup
Before working with Affymetrix® Microarray Suite, it is important to define where the
program stores and looks for files.
1. Launch Microarray Suite from the workstation and select Tools °˜ Defaults°˜ File
Locations from the menu bar.
2. The File Locations window displays the locations of the following files:
3. Verify that all three file locations are set correctly and click OK.
Contact Affymetrix Technical Support if you have any questions regarding this
procedure. To wash, stain, and scan a probe array, an experiment must first be defined in Microarray Suite.
1. Select Run °˜ Experiment Info from the menu bar. Alternatively, click the New
Experiment icon on the tool bar. The Experiment Information dialog box appears allowing the experiment name to be defined along with several other parameters, such as probe array type, sample description, and comments.
2. Type in the Experiment Name.
3. In the Probe Array Type box, click the arrow and select the probe array type from the drop-down list. Experiment name and probe array type are required. Complete as much of the other information as desired. The protocol information at the bottom of the dialog box is exported to the experiment information dialog box after the hybridization and scan are completed.
4. Save the experiment by selecting Save.
The name of the experiment is used by Microarray Suite to access the probe array type and data for the sample while it is being processed. Data files generated for the sample are automatically labeled to correspond to the experiment name. Microarray Suite automatically fills in the Protocol section of this dialog box with information on array processing from the fluidics station.
5. Close the Experiment Information dialog box.
SECTION 2 Eukaryotic Sample and Array Processing 2.4.8
The Fluidics Station 400, 450, or 250 is used to wash and stain the probe arrays. It is
operated using Microarray Suite.
Setting Up the Fluidics Station
1. Turn on the Fluidics Station using the toggle switch on the lower left side of the
machine.
2. Select Run °˜ Fluidics from the menu bar.
The Fluidics Station dialog box appears with a drop-down list for selecting the
experiment name for each of the fluidics station modules. A second drop-down list
is accessed for choosing the Protocol for each of the fluidics station modules.
Priming the Fluidics Station Priming ensures that the lines of the fluidics station are filled with the appropriate buffers and the fluidics station is ready for running fluidics station protocols.
Priming should be done:
1. To prime the fluidics station, select Protocol in the Fluidics Station dialog box.
2. Choose Prime or Prime_450 for the respective modules in the Protocol drop-down list.
3. Change the intake buffer reservoir A to Non-Stringent Wash Buffer, and intake buffer reservoir B to Stringent Wash Buffer.
4. Click Run for each module to begin priming.
Refer to the Fluidics Station User’s Guide for instructions on connecting and
addressing multiple fluidics stations.
CHAPTER 4 Eukaryotic Arrays: Washing, Staining, and Scanning 2.4.9
Eukaryotic
Probe Array Wash and Stain
Affymetrix offers two staining protocols: 1) the single stain protocol for eukaryotic targets (page 2.4.9), and 2) a signal amplification protocol for eukaryotic targets (page 2.4.13). Please use the Antibody Amplification Washing and Staining Protocol for all arrays with probe cells of 24 µm or smaller.
1. After 16 hours of hybridization, remove the hybridization cocktail from the probe array and set it aside in a microcentrifuge vial. Store on ice during the procedure or at -80°C for long-term storage.
2. Fill the probe array completely with the appropriate volume of Non-Stringent Wash
Buffer (Wash A), as given in Table 2.3.2 on page 2.3.8.
Preparing the SAPE Stain Solution
Streptavidin Phycoerythrin (SAPE) should be stored in the dark at 4°C, either foil-wrapped or kept in an amber tube. Remove SAPE from refrigerator and tap the tube to mix well before preparing stain solution. Do not freeze SAPE. Always prepare the SAPE stain solution immediately before use.
For each probe array to be stained, combine the following components in a microcentrifuge vial:
If necessary, at this point, the probe array can be stored at 4°C for up to 3 hours
before proceeding with washing and staining. Equilibrate the probe array to room
temperature before washing and staining.
Volumes needed will be the same for all fluidic protocols. This procedure takes
approximately 75 minutes to complete.
CHAPTER 4 Eukaryotic Arrays: Washing, Staining, and Scanning 2.4.13
Eukaryotic
This protocol is recommended for use with probe arrays with probe cells of 24 µm or
smaller. This procedure takes approximately 90 minutes to complete.
Preparing the Staining Reagents
Prepare the following reagents. Volumes given are sufficient for one probe array.
SAPE Stain Solution (see Table2.4.5 for SAPE solution mix)
Streptavidin Phycoerythrin (SAPE) should be stored in the dark at 4°C, either foil-wrapped or kept in an amber tube. Remove SAPE from refrigerator and tap the tube to mix well before preparing stain solution. Do not freeze SAPE. Always prepare the SAPE stain solution immediately before use. Mix well and divide into two aliquots of 600 µL each to be used for stains 1 and 3, respectively.
Antibody Solution (see Table2.4.6 for Antibody solution mix)
SECTION 2 Eukaryotic Sample and Array Processing 2.4.14
If you are using the Fluidics Station 450/250:
Washing and Staining the Probe Array
1. In the Fluidics Station dialog box on the workstation, select the correct experiment
name from the drop-down Experiment list.
The Probe Array Type appears automatically.
2. In the Protocol drop-down list, select the appropriate antibody amplification protocol
to control the washing and staining of the probe array format being used.
3. Choose Run in the Fluidics Station dialog box to begin the washing and staining.
Follow the instructions in the LCD window on the fluidics station.
If you are unfamiliar with inserting and removing probe arrays from the fluidics station modules, please refer to the appropriate Fluidics Station User’s Guide, or Quick Reference Card (P/N 08-0093 for the FS-450/250 fluidics stations).
4. Insert the appropriate probe array into the designated module of the fluidics station while the cartridge lever is in the, down, or eject, position. When finished, verify that the cartridge lever is returned to the up, or engaged, position.
See Table 2.4.7 for Fluidics protocol- Antibosy amplification for Eukaryotic Targets
SECTION 2 Eukaryotic Sample and Array Processing 2.4.16
If you are using the Fluidics Station 400:
Washing and Staining the Probe Array
1. In the Fluidics Station dialog box on the workstation, select the correct experiment
name in the drop-down Experiment list. The probe array type will appear
automatically.
2. In the Protocol drop-down list, select the appropriate antibody amplification protocol to control the washing and staining of the probe array format being used:
3. Choose Run in the Fluidics Station dialog box to begin the washing and staining.
Follow the instructions on the LCD window on the fluidics station.
4. If you are unfamiliar with inserting and removing probe arrays from the fluidics station modules, please refer to the Fluidics Station 400 User’s Guide, Fluidics Station 400 Video In-Service CD (P/N 900374), or Quick Reference Card (P/N 08-0072).
5. Insert the appropriate probe array into the designated module of the fluidics station while the cartridge lever is in the EJECT position. When finished, verify that the
cartridge lever is returned to the ENGAGE position.
6. Remove any microcentrifuge vial remaining in the sample holder of the fluidics station module(s) being used.
7. When the LCD window indicates, place the microcentrifuge vial containing 600 µL of streptavidin phycoerythrin (SAPE) stain solution into the sample holder. Verify that the metal sampling needle is in the vial with its tip near the bottom.
8. When the LCD window indicates, replace the microcentrifuge vial containing the
streptavidin stain with a microcentrifuge vial containing antibody stain solution into the sample holder, making sure that the metal sampling needle is in the vial with its tip near the bottom.
9. When the LCD window indicates, replace the microcentrifuge vial containing the
antibody stain solution with a microcentrifuge vial containing 600 µL of streptavidin
phycoerythrin (SAPE) stain solution into the sample holder. Verify that the metal
sampling needle is in the vial with its tip near the bottom.
The Fluidics Station dialog box and the LCD window display the status of the
washing and staining as they progress. When the wash is complete, the LCD
window displays the message EJECT CARTRIDGE.
10. At the end of the run, or at the appropriate prompt, remove microcentrifuge vial
containing stain and replace with an empty microcentrifuge vial.
11. Remove the probe arrays from the fluidics station modules by first moving the probe array holder lever to the EJECT position.
12. Check the probe array window for large bubbles or air pockets.
ENGAGE wash block and proceed to Probe Array Scan on page 2.4.18.
If you do not scan the arrays right away, keep the probe arrays at 4°C and in the dark
until ready for scanning. If there are no more samples to hybridize, shut down the fluidics station following the procedure outlined in the section, Shutting Down the Fluidics Station on page 2.4.20. For proper cleaning and maintenance of the fluidics station including the bleach protocol, refer to Section 4, Fluidics Station | | remark: | | | last_update: | 2003-09-05 09:37:20 | | login_id: | caldo | | resource: | Affymetrix manual |
| 2/2 | id: | 5 | | procedure_name: | Maizearray.org procedure for washing microarrays | | instrument: | | | software: | other | | description: | First wash in 2 X SSC, 0.5% SDS at 55 degrees with 30 sec flow, 1 1/2 min. hold, 2 cycles.
Second wash in 0.5 X SSC at 26 degrees as above.
Third wash in .05 X SSC as above. | | remark: | | | last_update: | 2005-10-11 10:57:46 | | login_id: | HakeOligo | | resource: | other |
|