|
| Status |
Public on Oct 28, 2007 |
| Title |
B6NTacAce.vs.B6J_rep1_hyb1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Bgl II genomic representation of DNA isolated from the liver of a C57BL/6NTacAce inbred mouse
|
| Organism |
Mus musculus |
| Characteristics |
DNA prep ID: C57BL/6NTacAce-L
Strain: C57BL/6NTacAce
Tissue: liver
Age: 4-8 weeks
Gender: Female
|
| Biomaterial provider |
Ace Animals, Inc.
|
| Growth protocol |
DNA was isolated using the Puregene Kit (Gentra Systems), with the following modifications: livers were flash frozen in liquid nitrogen, ground with mortar and pestle, and dounce-homogenized in 20 ml cell lysis solution. Two additional extrections were performed with phenol/chloroform/isoamyl alcohol, and chloroform/isoamyl alcohol, and DNA was precipitated with 2 volumes isopropanol and 1/10 volume sodium acetate.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was isolated using the Puregene Kit (Gentra Systems), with the following modifications: livers were flash frozen in liquid nitrogen, ground with mortar and pestle, and dounce-homogenized in 20 ml cell lysis solution. Two additional extrections were performed with phenol/chloroform/isoamyl alcohol, and chloroform/isoamyl alcohol, and DNA was precipitated with 2 volumes isopropanol and 1/10 volume sodium acetate.
1.5 µg of DNA was digested with the Bgl II restriction enzyme for 8-12 hrs at 37°C. Fragments were column-purified (Qiagen Minielute kit), and universal adaptors were added to fragments by overnight ligation with T4 ligase at 16º C. After 22 cycles of PCR amplification with the universal primer, resulting genomic representations were predominantly composed of fragments 100-1200 bp in length. Representations were then column-purified (Qiagen PCR purification kit).
|
| Label |
Cy5
|
| Label protocol |
1.5 µg of DNA from genomic representations were differentially labeled with Cy5-dCTP or Cy3-dCTP dyes using the Amersham-Pharmacia Megaprime kit. Labeled DNA was purified with Microcon columns (Millipore) with lowTE.
|
| |
|
| Channel 2 |
| Source name |
Bgl II genomic representation of DNA isolated from the liver of a C57BL/6J inbred mouse
|
| Organism |
Mus musculus |
| Characteristics |
DNA prep ID: C57BL/6J-1-L
Strain: C57BL/6J
Tissue: liver
Age: 4-8 weeks
Gender: Female
|
| Biomaterial provider |
The Jackson Laboratory
|
| Growth protocol |
DNA was isolated using the Puregene Kit (Gentra Systems), with the following modifications: livers were flash frozen in liquid nitrogen, ground with mortar and pestle, and dounce-homogenized in 20 ml cell lysis solution. Two additional extrections were performed with phenol/chloroform/isoamyl alcohol, and chloroform/isoamyl alcohol, and DNA was precipitated with 2 volumes isopropanol and 1/10 volume sodium acetate.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was isolated using the Puregene Kit (Gentra Systems), with the following modifications: livers were flash frozen in liquid nitrogen, ground with mortar and pestle, and dounce-homogenized in 20 ml cell lysis solution. Two additional extrections were performed with phenol/chloroform/isoamyl alcohol, and chloroform/isoamyl alcohol, and DNA was precipitated with 2 volumes isopropanol and 1/10 volume sodium acetate.
1.5 µg of DNA was digested with the Bgl II restriction enzyme for 8-12 hrs at 37°C. Fragments were column-purified (Qiagen Minielute kit), and universal adaptors were added to fragments by overnight ligation with T4 ligase at 16º C. After 22 cycles of PCR amplification with the universal primer, resulting genomic representations were predominantly composed of fragments 100-1200 bp in length. Representations were then column-purified (Qiagen PCR purification kit).
|
| Label |
Cy3
|
| Label protocol |
1.5 µg of DNA from genomic representations were differentially labeled with Cy5-dCTP or Cy3-dCTP dyes using the Amersham-Pharmacia Megaprime kit. Labeled DNA was purified with Microcon columns (Millipore) with lowTE.
|
| |
|
| |
| Hybridization protocol |
Hybs were performed in 25 uL of solution consisting of 37.5% formamide, 4x SSC, and O.1% SDS. Samples were denatured at 95° for 5 min, pre-annealed for 30 min at 37° with 1 µg of mouse COT1 DNA, applied to the microarray, and hybridized under a coverslip at 42°C for 14-18 h. Slides were washed for 1 min in 0.2% SDS/0.2x SSC, 30 sec in 0.2x SSC, 30 sec in 0.05x SSC, and dried by centrifugation.
|
| Scan protocol |
Microarrays were scanned with an Axon GenePix 4000B scanner with pixel size set to 5um. GenePix Pro 4.0 software was used for gridding and quantitation of intensities.
|
| Description |
none
|
| Data processing |
Measured intensities without background subtraction were used to calculate ratios. Data were normalized using an intensity-based lowess curve fitting algorithm similar to that described in Yang et al (Nucleic Acids Research, 30 e15, Feb 15, 2002).
|
| |
|
| Submission date |
Sep 07, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Ira M Hall |
| E-mail(s) |
hall@cshl.edu
|
| Phone |
516-287-3764
|
| Organization name |
Cold Spring Harbor Laboratory
|
| Lab |
Hall
|
| Street address |
1 Bungtown Road
|
| City |
Cold Spring Harbor |
| State/province |
NY |
| ZIP/Postal code |
11724 |
| Country |
USA |
| |
|
| Platform ID |
GPL5824 |
| Series (1) |
| GSE8980 |
Recurrent DNA copy number variation in the laboratory mouse |
|
