| 1/4 | id: | 5 | | login_id: | caldo | | protocol_name: | Affy- Eukaryotic Target Hybridization | | chamber_type: | Hybridization Oven 640 | | quantity: | 15 | | duration: | 16 | | volume: | 250 | | temperature: | 45 | | description: | SECTION 2 Eukaryotic Sample and Array Processing 2.3.6
Reagent Preparation
12X MES Stock
(1.22M MES, 0.89M [Na+])
For 1,000 mL:
70.4g MES-free acid monohydrate
193.3g MES Sodium Salt
800 mL of Molecular Biology Grade water
Mix and adjust volume to 1,000 mL.
The pH should be between 6.5 and 6.7. Filter through a 0.2 µm filter.
2X Hybridization Buffer
(Final 1X concentration is 100 mM MES, 1 M [Na+], 20 mM EDTA, 0.01% Tween 20)
For 50 mL:
8.3 mL of 12X MES Stock
17.7 mL of 5 M NaCl
4.0 mL of 0.5 M EDTA
0.1 mL of 10% Tween 20
19.9 mL of water
Store at 2°C to 8°C, and shield from light
Do not autoclave. Store at 2°C to 8°C, and shield from light.
Discard solution if yellow.
CHAPTER 3 Eukaryotic Target Hybridization 2.3.7
Eukaryotic
Eukaryotic Target Hybridization
Please refer to the table below for the necessary amount of cRNA for appropriate probe array format. These recipes take into account that it is necessary to make extra hybridizationcocktail due to a small loss of volume (10-20 µL) during each hybridization.
1. Mix the following for each target, scaling up volumes for hybridization to multiple
probe arrays. See Table 2.3.1 in Affy manual
2. Equilibrate probe array to room temperature immediately before use.
3. Heat the hybridization cocktail to 99°C for 5 minutes in a heat block.
4. Meanwhile, wet the array by filling it through one of the septa (see for
location of the probe array septa) with appropriate volume 1X Hybridization Buffer
using a micropipettor and appropriate tips.
5. Incubate the probe array filled with 1X Hybridization Buffer at 45°C for 10 minutes
with rotation. It is imperative that frozen stocks of 20X GeneChip Eukaryotic Hybridization Control is heated to 65°C for 5 minutes to completely resuspend the cRNA before aliquotting. It is important to allow the arrays to equilibrate to room temperature completely. Specifically, if the rubber septa are not equilibrated to room temperature, they may be prone to cracking, which can lead to leaks. It is necessary to use two pipette tips when filling the probe array cartridge: one for filling and the second to allow venting of air from the hybridization chamber.
6. Transfer the hybridization cocktail that has been heated at 99°C, in step 3, to a 45°C heat block for 5 minutes.
7. Spin hybridization cocktail(s) at maximum speed in a microcentrifuge for 5 minutes to remove any insoluble material from the hybridization mixture.
8. Remove the buffer solution from the probe array cartridge and fill with appropriate
volume ( on page 2.3.8) of the clarified hybridization cocktail, avoiding any insoluble matter at the bottom of the tube.
9. Place probe array into the Hybridization Oven, set to 45°C. Avoid stress to the motor; load probe arrays in a balanced configuration around the axis. Rotate at 60 rpm.
10. Hybridize for 16 hours.
During the latter part of the 16-hour hybridization, proceed to Section 2, Chapter 4 to prepare reagents required immediately after completion of hybridization. | | remark: | | | last_update: | 2003-09-17 09:49:45 | | resource: | Affymetrix manual | | quantity_unit: | ug | | duration_unit: | hrs | | volumn_unit: | ul | | temperature_unit: | C |
| 2/4 | id: | 15 | | login_id: | da_xia | | protocol_name: | Hybridization with cDNA targets - Spotted NSF Maize Oligo Array | | chamber_type: | other | | quantity: | 0 | | duration: | 0 | | volume: | 0 | | temperature: | 0 | | description: | Hybridization with cDNA targets
Overview
This protocol describes our microarray hybridization and washing procedures for cDNA targets
and has been optimized to produce images with a high signal to noise ratio. This protocol is
based on a 2X SSC, non formamide hybridization buffer and works well with cDNA target
prepared using direct or indirect labeling methods. While the protocol below works for cRNA
targets, it does result in a higher background and for this reason we are recommending an
alternative protocol for cRNA targets which is posted at www.maizearrays.org. The Telechem
hybridization cassettes and washing station are easy to use and produce consistent results though
hybridization cassettes and washing stations produced by other manufacturers would work as
well. Microarray hybridization and washing are critical steps in the overall process of obtaining
consistent expression data values and one or more practice runs using the procedure below are
advised before scaling up to do larger numbers of hybridizations.
Materials
Microarray holder and wash station (Telechem International Cat# HTW)
10 mL disposable pipette
50 mL tubes
Sterile measuring cylinder
Extra-deep Hybridization Cassettes (Telechem International Cat# AHCXD)
LifterSlip (Erie Scientific Cat#24X60I-2-4733, ordered through VWR or Fisher Scientific)
Hybridization oven set to 55°C
Liquid Blocking Reagent (Amersham; Cat # RPN3601)
2% SDS
20XSSC (Invitrogen Ultrapure, Cat# 15557-044)
DEPC treated H2O
Procedure
DNA Probe Immobilization:
Re-hydration and UV cross linking can be done well in advance before microarray hybridization,
and the slides can be stored at room temperature for several months. However we do not
recommend storage of washed microarray slides for extended periods of time.
1. Mark the corner boundaries of the array on a separate glass slide.
a. Once spots have been immobilized and the slide is washed, the spots will not be
visible (the spots are only visible due to the presence of SSC crystals).
b. One needs to know the boundaries of the array in order to correctly place coverslip
over array during hybridization.
2. Re-hydrate slide over a 50°C water bath for 5 seconds.
a. Hold slide label side down over the water vapor.
b. Watch spots carefully so that they do not over-hydrate and begin to merge together.
In humid environments this is particularly important.
3. Snap dry the slide on a 45°C heating block for 5 seconds.
a. Place slide label side up on heating block.
b. Allow slide to cool for 1 minute or cool with compressed air by blowing over the
back of the slide.
4. Repeat steps 1-3 a total of four times.
1
5. UV cross-link the slides by exposing them in batches, label side up, to 180 mJ in a
commercial cross-linker (we employ a Stratalinker).
6. Wash the slide in 1% SDS for 5 minutes at RT on a shaker or agitate by hand.
7. Remove SDS by dipping the slides ten times into DDH2O.
8. Immediately transfer the slides to 100% ethanol, dip five times, then incubate for three
minutes with shaking.
9. Spin dry slide in centrifuge at no more than 200 x g for 2-4 minutes.
a. Pack bottom of 50 mL centrifuge tube with Kimwipes.
b. Using forceps carefully place slide into tube with label at the bottom.
c. Repeat spin if any liquid is remaining on the slide surface.
10. Repeat the ethanol wash if any visible streaks remain after spin dry.
11. Store slide in a lint-free light-proof box at RT with low humidity.
Hybridization Setup:
Hybridization Mix (160 µl is enough to hybridize both the MOA and MOB slides)
1. Mix the following in a microfuge tube:
20X SSC 16.0 µl
Liquid Block 4.8 µl
2% SDS 6.4 µl
Both Labeled Targets* µl
H2O to 150 µl
*We have obtained the most consistent results using 2-3 ug of each target per slide. Therefore, a
160 µl hybridization mix which can be hybridized to both the MOA and MOB slides will have 4
to 6 ug of each Cy labeled target. It may be necessary to optimize the number of ug of target on
each array to your individual laboratory setting but the above values provide a good starting
point. We have found that loading by picomoles of target often produces unbalanced images.
2. Denature labeled target by incubating tube at 65o C for 5 min.
3. Apply on to the slides directly (preferred) or place on ice.
4. Rinse ArrayItâ„¢ Hybridization Cassette with distilled water and dry thoroughly.
5. Make sure flexible rubber gasket is seated evenly in gasket channel.
6. Add 15. µl water to the lower groove inside the cassette chamber.
7. Insert the microarray (1" x 3" or 25mm x 75mm slide) into cassette chamber, DNA side up.
8. Place the lifter slip over the microarray slide (make sure the white stripe of the lifterslip is at
the lower side)
9. Apply the PRE-HEATED sample slowly to the one end of the lifterslip and let it disperse.
Use 60 to 70µl of sample for each slide being careful not to introduce air bubbles.
10. Quickly place the clear plastic cassette lid on top of the cassette chamber.
11. Apply downward pressure and manually tighten (clockwise) the four sealing screws.
12. Check all four screws again to confirm a tight seal.
13. Place the cassette into a hybridization oven set at 55C.
14. Allow the hybridization reaction to proceed for 12 to 14 hours.
15. After hybridization, remove cassette, manually loosen the four sealing screws
(counterclockwise) and remove lid.
2
16. Remove the microarray slide from the cassette chamber using forceps and place the slides
into the washing buffer.
Microarray Washing
1. Wash slide in the following solutions for 5 min each:
2x SSC, 0.1% SDS @ 55°C
0.5x SSC @ RT (second wash)
0.1x SSC @ RT (third wash)
2. Washing is done by immersing the slides in a glass the Telechem wash station (Cat# HTW)
containing approximately 400 ml of wash buffer, followed by placing it on a magnetic stir
plate set at ~120 rpm. Pre-heat the first wash solution to 55°C, and make sure the slides are
completely immersed in wash buffer. To ensure even washing, rotate the slide holder 90
degrees mid way through each wash.
3. After completion of the washes, plunge the slide holder five times in RT 0.1X SSC and spin
dry the slide in the centrifuge at no more than 1000 rpm for 2-4 min.
a. Pack bottom of 50 mL plastic disposable centrifuge tube with Kimwipes.
b. Using forceps, carefully place slide into tube with label at the bottom.
c. Repeat spin if any liquid remains on the slide.
Note: Washing is a critical step and care needs to be taken not to over or under wash your
slides. For consistent results it is advisable to wash the slides the same way each time.
4. Scan slide immediately, or store in a light proof box @ room temp under dry conditions.
Save the image as .TIFF file. Immediate scanning is recommended. However, we have
observed that properly stored slides (light protected-dry- RT) can retain the signal up to a
month. Some reports indicate environmental pollutants (ozone) can drastically affect
fluorescence. Examine the scanned images immediately to determine the number of
elements that are near zero or are saturated (for a 16-bit scanner, this represents a value of
65,400). The proportion of these elements should be acceptably low, since information is
lost in either case. It is much more preferable to rescan with altered gain settings on the
scanner than to proceed with analysis of images containing large proportions of zero or
saturated elements.
| | remark: | | | last_update: | 2005-10-04 07:51:01 | | resource: | | | quantity_unit: | ug | | duration_unit: | hrs | | volumn_unit: | ul | | temperature_unit: | C |
| 3/4 | id: | 16 | | login_id: | HakeOligo | | protocol_name: | protocol from Maize Oligonucleotide Array Project
| | chamber_type: | | | quantity: | 3 | | duration: | 0 | | volume: | 0 | | temperature: | 50 | | description: | For hyb solution, conditions are used, and washing, I
followed the protocol from Maize Oligonucleotide Array Project
(http://www.maizearray.org/). For determination of labeled polynucleotide
input, I used the protocol from CSHL facility (Attached file: hybrid. pdf).
Although there is one mistake in that file as below. (page 3 line5)
Cy3 Combination Ratio=Cy3 FOI/ (Cy3 FOI + Cy5 FOI)
should be
Cy3 Combination Ratio=1-(Cy3 FOI/ (Cy3 FOI + Cy5 FOI))
| | remark: | | | last_update: | 2005-10-07 14:26:59 | | resource: | | | quantity_unit: | ug | | duration_unit: | hrs | | volumn_unit: | ul | | temperature_unit: | C |
| 4/4 | id: | 17 | | login_id: | HakeOligo | | protocol_name: | protocol from Maize Oligonucleotide Array Project | | chamber_type: | other | | quantity: | 50 | | duration: | 14 | | volume: | 100 | | temperature: | 55 | | description: | Hybridization:
Mix:
20 X SSC 25 ul
Liquid Block 15 ul
2% SDS 10 ul
Labeled Targets 80 ul each
H2O 40 ul
Heat 2 min. 99 degrees in thermocycler, transfer to ice.
Load 100 ul on each slide on GeneTac Hyb. Station.
Incubate 55 degrees, 14 hrs. with agitation of the probe.
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 10:50:24 | | resource: | | | quantity_unit: | ug | | duration_unit: | hrs | | volumn_unit: | ul | | temperature_unit: | C |
|