Biomedical Engineering Reference
In-Depth Information
Notes
n
This blocking solution is specific to the blocking of CodeLink
™
slides (SurModics Inc) on which
amino linker-containing oligonucleotides (dissolved at 10
μ
M in 50 mM sodium phosphate buffer
pH 8.5) are spotted.
o
Extend to 30min if the blocking solution is not pre-warmed, but do not exceed 1 h.
p
Use at least 10ml of wash solution per slide.
q
Test and reference DNA can be labeled with either Cy3 or Cy5.
r
If many experiments are planned, we recommend ordering a large batch of Cot-1 DNA from the
same lot.
s
Take care to prevent foam formation due to the SDS.
t
Alternative manual protocol for hybridization and washing: cut off the large end of a 200
μ
l
pipette tip to fit on a 5ml syringe and fill the syringe with rubber cement (Ross). Apply
the rubber cement closely around an array. Apply a second or third layer of rubber cement
thickly. Apply the hybridization mixture to the array and incubate the slide assembly in a
closed incubation chamber over two nights at 37
◦
C on a rocking table. Following hybridization,
disassemble the slide assembly and rinse the hybridization solution from the slide in a room
temperature stream of PN buffer. Wash the array in wash buffer for 10-15 min at 45
◦
C, followed
by a 10-15min room temperature wash in PN buffer. Carefully remove the rubber cement (do
not let the array dry) with tweezers/forceps, wash the array sequentially with 0.2
×
SSC and
0.1
×
SSC and centrifuge dry (250g, 3 min).
u
Cleaning the hybstation after each hybridization is essential to maintain proper functioning
of the equipment.
PROTOCOL 1.7 Scanning and Creation of a Copy
Number Profile
Equipment and reagents
•
High-resolution laser scanner, or imager equipped to detect Cy3 and Cy5 dyes,
including software to acquire images (e.g. Microarray Scanner G2505B, Agilent
Technologies)
•
Feature-extraction software (e.g. Bluefuse 3.2 (BF), BlueGnome Ltd, UK)
•
Gene array list (GAL-file, or equivalent) - created by the microarray printer software
using the oligonucleotide plate content lists provided by the supplier of the oligo
library
Position list: a file, containing the relative positions of the oligonucleotides in the
genome under investigation, provided by the supplier of the oligo library, or created by
mapping the oligonucleotide sequences onto the genome concerned
•
Software which calculates ratios, links the genomic position of the oligonucleotide to
the experimental ratios and draws a profile (e.g. Microsoft Excel, or dedicated software
such as BF).
•
