John M. S. Bartlett.pdf - Bio-Nica.info

250 Ringquist et al.
3. Purification of fluorescently labeled material from unincorporated dye was performed by
centrifuging the sample in a microfuge using Microcon columns twice, using 500 µL TE
(pH 8.0) washes each time, according to the manufacturer’s instructions.
4. Labeling of PCR products can also be performed by Terminal Transferase-catalyzed
addition of Cy3-dCTP or Cy5-dCTP, as well as other fluorescent-labeled or modified
deoxynucleotide triphosphates. 1 µg of PCR product in 20 µL was combined with
25 µL 2× terminal transferase buffer and 5 µL of 1 mM Cy5-dCTP or 1 mM Cy3-dCTP.
The reaction was performed at 37°C for 1 h and purified using the Microcon columns
as described previously.
5. After purification over microcon columns, fluorescent probe was dried under vacuum and
dissolved in 8 µL of 1× TE buffer. Preparation of probe in hybridization solution was
performed by addition of 8 µL of probe solution to 2 µL of blocking solution, 2.1 µL of
20× SSC, and 0.4 µL of 10% SDS. The solution was then incubated 1 min in a boiling
water bath, and allowed to cool on the bench top for 30 min.
3.6. Microarray Hybridization
1. Preparation of microarrays followed the protocol of Eisen and Brown (16) and used
polylysine-treated slides and PCR products generated from sequenced human cDNA
libraries; please see Eisen and Brown (16) for details on preparing polylysine-coated slides.
Inserts from the IMAGE library, supplied by Research Genetics, were PCR amplified by
standard means using the vector-specific primers. Printing of PCR products was done
using an Omnigrid robot to spot PCR products onto GoldSeal glass slides using a 16 pin
print head. After printing, slides were rehydrated briefly over an 80°C waterbath and snap
heated for about 6 s by placing the slides on an 80°C heat block. Ultraviolet treatment was
performed using a Stratalinker 2400 (Stratagene, La Jolla, CA) at 60 mJ for 1 min. The
slides were then baked at 80°C for 2 h and stored.
2. Immediately before hybridization, slides were treated with succinic anhydride as described
by Eisen and Brown (16). The succinic anhydride blocking solution must be prepared
fresh by placing 6 g of succinic anhydride into a dry beaker followed by addition of
335 mL of 1-methyl-2-pyrrolidinone. After the succinic anhydride was dissolved 15 mL
of 1 M sodium borate, pH 8.0 was added. The solution was stirred for 5 s to mix the
solutions. The mixture was then poured into a clean glass chamber and the slides placed
quickly inside and incubated for 15 min.
3. Immediately after succinic anhydride blocking, slides were placed in a chamber with
boiling water (the volume should be at least twice that of the blocking solution). The slides
were plunged up and down and shaken for 2 min, and then the slides were immediately
transferred to a chamber containing 95% ethanol, agitated and then dried for 37°C for
15 min.
4. Hybridization was performed by placing one glass chamber with distilled water in a 65°C
oven for at least 30 min before doing the hybridization. One hybridization chamber was
cleaned with pressurized air, and 2 drops (10 µL) of 3XSSC (450 mM sodium chloride,
45 mM sodium citrate, pH 7.0) were placed in the wells. The slides were then cleaned with
pressurized air and placed in the hybridization chamber. The probe was then pipetted onto
the slide, usually in 40 µL, taking care to avoid bubbles. The coverslip was then cleaned
with pressurized air and placed over the probe. This volume of probe usually spreads easily
along the surface; if not, the overslip can be pressed to help the spreading. The chamber
was then sealed and submerged in a 65°C water bath for 4 to 16 h.
5. The hybridization was stopped by washing in 2× SSC, 0.1% SDS, followed by washing
in 1× SSC. Samples were incubated for 2 min with 0.2× SSC. The washing steps were
performed at room temperature. Drying should be avoided in all the washing steps. Prepare