9. Wash again by adding 200 µl TE pH 7.5 and concentrating to about 20-30 µl (approximately 8-10 min at 500 x g).
Alternatively, a smaller pore MicroCon 30 can be used to speed the concentration step. In this case, centrifuge the first wash for approximately 4.5 minutes at 16,000 xg and the second (200 µl wash) for about 2.5 minutes at 16,000 xg.
10. Recover by inverting the concentrator over a clean collection tube and spinning for 3 min at 500 x g.
In some cases, the cy5 labeled cDNA will form a gelatinous blue precipitate that is recovered in the concentrated volume. The presence of this material signals the presence of contaminants. The more extreme the contamination, the greater the fraction of cDNA which will be captured in this gel. Even if heat solubilized, this material tends to produce uniform, non-specific binding to the DNA targets.
When concentrating by centrifugal filtration, the times required to achieve the desired final volume are variable. Overly long spins can remove nearly all the water from the solution being filtered. When fluor-tagged nucleic acids are concentrated onto the filter in this fashion, they are very hard to remove, so it is necessary to approach the desired volume by conservative approximations of the required spin times. If control of volumes proves difficult, the final concentration can be achieved by evaporating liquid in the speed-vac. Vacuum evaporation, if not to dryness, does not degrade the performance of the labeled cDNA.
11. Take a 2-3 µl aliquot of the Cy5 labeled cDNA for analysis, leaving 18-28 µl for hybridization.
12. Run this probe on a 2% agarose gel (6cm wide x 8.5 cm long, 2 mm wide teeth) in Tris Acetate Electrophoresis Buffer (TAE).
For maximal sensitivity when running samples on a gel for fluor analysis, use loading buffer with minimal dye and do not add ethidium bromide to the gel or running buffer.
13. Scan the gel on a Molecular Dynamics Storm fluorescence scanner (setting: red fluorescence, 200 micron resolution, 1000 volts on PMT)
Successful labeling produces a dense smear of probe from 400 bp to >1000 bp, with little pile-up of low molecular weight transcripts (as in Figure 1, Lane A). Weak labeling and significant levels of low molecular weight material indicates a poor labeling (as in Figure 1, Lane B). A fraction of the observed low molecular weight material is unincorporated fluor nucleotide.
Hybridization
5.1 Hybridize fluorescent cDNA to slide
1. Determine the volume of hybridization solution required. The rule of thumb is to use 0.033 µl for each mm2 of slide surface area covered by the cover slip used to cover the array. An array covered by a 24 mm by 50 mm coverslip will require 40 µl of hybridization solution. The volume of the hybridization solution is critical. When too little solution is used, it is difficult to seat the coverslip without introducing air bubbles over some portion of the arrayed ESTs, and the coverslip will not sit at a uniform distance from the slide. If the coverslip is bowed toward the slide in the center, there will be less labeled cDNA in that area and hybridization will be non-uniform. When too much volume is applied, the coverslip will move easily during handling, leading to misplacement relative to the arrayed ESTs, and non-hybridization in some areas of the array.
2. For a 40 µl hybridization, pool the Cy3 and Cy5 labeled cDNAs into a single 0.2 ml thinwall PCR tube and adjust the volume to 30 µl by either adding DEPC H2O, or removing water in a SpeedVac. If using a vacuum device to remove water, do not use high heat or heat lamps to accelerate evaporation. The fluorescent dyes could be degraded.
3. For a 40 µl hybridization combine the following components:
High Sample Blocking
High Array Blocking
Cy5 Cy3 probe
30 µl
28 µl
Poly d(A) (8mg/ml)
1 µl
2 µl
Yeast tRNA (4mg/ml)
1 µl
2 µl
Human C0t-1 DNA (10mg/ml)
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