Analysis of Gene Regulatory Circuits - Microarray Technology and Its Applications - page 264

600250-51)] is added to the sample. Subsequently, the following PCR cycle is
performed:

step 1: 72 ◦ C for 5 minutes;
step 2: 95 ◦ C for 2 minutes;
step 3: 95 ◦ Cfor1minute;
step 4: 60 ◦ Cfor1minute;
step 5: 72 ◦ Cfor1minute;
step 6: go to step 3 for 22 times;
step 7: 72 ◦ C for 5 minutes;
step 8: 4 ◦ C forever;

Afterwards, the DNA is purified using the Qiaquick PCR purification kit
(Qiagen, cat# 28106) and eluted in 60 µ l elution buffer provided with the kit.

DNA Microarray Hybridization

Labelling Immunoprecipitated DNA. To 200 ng of DNA from the pre-
vious step, 20 random primer solution (from the BioPrime kit, In-

vitrogen, Cat# 18094-011) and dH2O are added to a final volume of 42.5 µ l.
The mixture is boiled for 5 minutes and then immediately placed on ice. To
initiate the labelling reaction, 5

µ lof2.5 ×

low dCTP mixture (2.5 mM each
for dATP, dTTP and dGTP, and 0.6 mM for dCTP), 1.5

µ lof10 ×

l of Cy5-dCTP
(Amersham, Cat# PA55021) or Cy3-dCTP (Amersham, Cat# PA53021), 40
unit of Klenow DNA polymerase are added to the mixture. The reaction is
carried out at 37 ◦ C for 2 hours. Finally, the labelled DNA is purified using
the Qiagen PCR kit (Qiagen, Cat# 28106).

µ

DNA Microarray Hybridization. 2.5 µ g of Cy5-labelled ChIP DNA,

2.5 g human Cot-1 DNA (In-

vitrogen, Cat# 15279-011) are mixed together and concentrated by ethanol
precipitation. The DNA pellet is dissolved in 22.4

µ g of Cy3-labelled genomic DNA and 36 µ

µ l of hybridization buffer 1.

Then 20 l of hybridization buffer 2 is added to the mixture, and the sample is

incubatedfirstat95 ◦ C for 5 minutes then 42 ◦ C for 2 minutes. Subsequently,
4

µ

µ l of yeast tRNA (Sigma, cat# R9001 at 10 µ g/ µ l) and 3 µ l of 2% BSA are

used to adjust the hybridization reaction to 50 l. This mixture is added to

a DNA microarray slide that has been incubated with the pre-hybridization
solution for 40 minutes at 42 ◦ C.A25mm

µ

60 mm cover slip is then gen-
tly placed on top of the sample, and the hybridization is carried out in a
hybridization chamber (Corning, cat# 07-200-271) at 60 ◦ C overnight in a
water bath.

×

Washing Microarrays. After the hybridization, the microarray slide is
washed once with washing buffer 1 at 60 ◦ C for 5 minutes, once with washing
buffer 2 for 10 minutes at room temperature, and three times with washing
buffer 3 at room temperature.