A white single colony of S. pombe cells on a YE plate was inoculated into YES liquid medium to grow to 5x(the 6th power of 10)cell/ml at 30°C.
Extracted molecule
genomic DNA
Extraction protocol
DNA isolated from immunoprecipitates was amplified according to methods in (Katou et al., 2003, Nature vol424, pp1078-1083). In the first round of amplification, A primer (GGAATTCCAGCTGACCACCNNNNNNNNN) and the template DNA were incubated in reaction mixture of Sequenase Ver2.0 T7 DNA polymerase (USB). In the second round of amplification, B primer (GGAATTCCAGCTGACCACC) and product of the first round was incubated in reaction mixture of Ex Taq polymerase (TAKARA).
Label
Cy5
Label protocol
The amplified DNA were labeled with Cy5-dUTP by Oligotailing kit (Rochediagnostics).
A white single colony of S. pombe cells on a YE plate was inoculated into YES liquid medium to grow to 5x(the 6th power of 10)cell/ml at 30°C.
Extracted molecule
genomic DNA
Extraction protocol
Genome DNA was isolated from the S.pombe cell nuclei according to Matsumoto et al., 1987, Mol. Cell. Biol. vol.7 pp 4424-4430. The isolated genomic DNA was amplified according to methods in (Katou et al., 2003, Nature vol424, pp1078-1083). In the first round of amplification, A primer (GGAATTCCAGCTGACCACCNNNNNNNNN) and the template DNA were incubated in reaction mixture of Sequenase Ver2.0 T7 DNA polymerase (USB). In the second round of amplification, B primer (GGAATTCCAGCTGACCACC) and product of the first round was incubated in reaction mixture of Ex Taq polymerase (TAKARA).
Label
Cy3
Label protocol
The amplified DNA were labeled with Cy3-dUTP by Oligotailing kit (Rochediagnostics).
Hybridization protocol
The labeled targets were added to the hybridization solution to contain an equal amount of the Cy3 and Cy5, and then were hybridized onto DNA on the microarray, using Genomic solutions GeneTac Hybridization Station, for four hours at 40°C in Genomic solutions GeneTac Hyb buffer 120microl (including 42% formamide ). The slides after hybridization were washed in the following sequence: (1) 2xSSC 0.1%SDS at 40°C for 5min, (2) 0.2xSSC at 25°C for 1min, and (3) 0.1xSSC at 25°C for 1min.
Scan protocol
Microarrays were scanned using an ArraywoRx arrayscanner, and the acquired data was analyzed by a SoftwoRx software (Applied Precision, Inc., Seattle, WA). Fluorescent intensity in each spot circle (150microm diameter) was measured by Softworx tracker.
Description
Comparing between genomic DNA (Cy3) and DNA from immunoprecipitates with anti Swi6(Cy5) of wild type haploid.
Data processing
Measured fluorescent intensity, I, was corrected as follows to give a corrected intensity, C:C = I-M, (for I>M+2s), C = I*2s/(M+2s), (for I<M+2s) where M and s are an average and a standard deviation of I of negative control spots for each wave length respectively. When I = M+2s, that is C=2s, it was set to be a detection limit. When C for either Cy3 or Cy5 or both were greater than 2s, the values were considered to be effective data. VALUE (Copy ratio r') of the each effective detection spot obtained thus was scaled as follows: r'=r-m, r=logR(base is 2), R=(Ccy5/Ccy3), m is an average of r of all effective detection spots.
Submission date
Jul 18, 2007
Last update date
Jan 23, 2023
Contact name
Atsushi Matsuda
Organization name
National Institute of Information and Communications Technology
