Philip R. Hardwidge,University of Kansas Medical Center, USA,James M. Fleckenstein,U. Tennessee Health Science Center, USA,Robert J. Belland,U. Tennessee Health Science Center , USA, Roy Curtiss, III,Arizona State University, USA, David H. Francis,South Dakota State University, USA, Oscar G. Gomez-Duarte University of Iowa Childrens' Hospital, USA,Robert P. Gormley,US Navy, USA, Rodney A. Moxley,University of Nebraska, USA, George P. Munson,University of Miami, USA Firdausi Qadri,Intl. Centre for Diarrhoeal Disease Research, Bangladesh, Stephen J. Savarino,US Navy, USA
Treatment protocol
None
Growth protocol
cells were grown in LB overnight at 37 degrees celcius.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was isolated by lysozyme and proteinase K treatment.
Label
Cy3
Label protocol
4ug of genomic DNA was labeled at 37 degree Celcius overnight using DNA polymerase I Klenow fragment (New England Biolabs), a 25X 2:1 amino allyl dUTP (Ambion) dNTP mix, and random hexamers (Invitrogen). Labeled DNA was purified with Qiaquick PCR purification kit (QIAGEN).The purified labelled DNA was then dye coupled with the appropriate cye dye (CY3/CY5).
Philip R. Hardwidge,University of Kansas Medical Center, USA,James M. Fleckenstein,U. Tennessee Health Science Center, USA,Robert J. Belland,U. Tennessee Health Science Center , USA, Roy Curtiss, III,Arizona State University, USA, David H. Francis,South Dakota State University, USA, Oscar G. Gomez-Duarte University of Iowa Childrens' Hospital, USA,Robert P. Gormley,US Navy, USA, Rodney A. Moxley,University of Nebraska, USA, George P. Munson,University of Miami, USA Firdausi Qadri,Intl. Centre for Diarrhoeal Disease Research, Bangladesh, Stephen J. Savarino,US Navy, USA
Treatment protocol
None
Growth protocol
cells were grown in LB overnight at 37 degrees celcius.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was isolated by lysozyme and proteinase K treatment.
Label
Cy5
Label protocol
4ug of genomic DNA was labeled at 37 degree Celcius overnight using DNA polymerase I Klenow fragment (New England Biolabs), a 25X 2:1 amino allyl dUTP (Ambion) dNTP mix, and random hexamers (Invitrogen). Labeled DNA was purified with Qiaquick PCR purification kit (QIAGEN).The purified labelled DNA was then dye coupled with the appropriate cye dye (CY3/CY5).
Hybridization protocol
Equal concentrations of DNA probe from the appropriate Cy3 and Cy5 labeled probes were combined, dried and then resuspended in hybridization buffer as defined by the Agilent Oligonucleotide Array-Based CGH protocol. Resuspended probes were denatured at 95 degree Celcius and held at 37 degree Celcius prior to hybridization. The probe mixture then was added to the Agilent microarray slide and allowed to hybridize for 24 hours rotating at 20 rpm in a hybridization oven at 65 degree Celcius. Hybridized slides were washed sequentially as defined by the Agilent Oligonucleotide Array-Based CGH protocol.
Scan protocol
Scanned on Axon GenePix 4000 scanner. PMT values were optimized during scanning to balance channel intensities.
Data processing
Procedures to calculate the value of log2 ratio:
1. Use Agilent software Feature Extraction to analyze spotted arrays.
2. Use TM4 suite software ExpressConverter to modify Agilent Feature Extraction file into *.mev.
3. Calculate the value of log2 ratio (Reference/Query) from the normalized .mev file:
3a). If both channels (QUERY_MEDIAN_INTENSITY and REF_MEDIAN_INTENSITY) are zero, assign the value "null".
3b). If one channel is zero and the other is not zero, substitute the zero with one (1) and then, assign the log2 ratio of QUERY_MEDIAN_INTENSITY/REF_MEDIAN_INTENSITY to VALUE
3c). If neither channel is zero, assign the log2 ratio of QUERY_MEDIAN_INTENSITY/REF_MEDIAN_INTENSITY to VALUE.
Novel gene discovery in up to 10 ETEC strains and production of species microarray. Discovery of conserved and unique ETEC genes by comparative genome hybridization studies.
Data table header descriptions
ID_REF
VALUE
log2 ratio of QUERY_MEDIAN_INTENSITY/REF_MEDIAN_INTENSITY.
