1. Grow overnight culture of Streptococcus uberis in Todd Hewitt Broth at 37C 2. Pipet 1ml of culture into 1.7ml microfuge tube 3. Pellet cells at 13,000-16,000g for 15 seconds. Discard supernatant 4. Add 300ul of Genomic DNA lysis Solution and pipet up and down to suspend 5. Add 1.5ul of 4,000u/ml lysozyme (up to 10,000u/ml) 6. Incubate at 80C for 5 minutes to lyse cells 7. Add 1.5ul RNase solution (4mg/ml) to lysate 8. Mix by invertin 25 times and incubate at 37C for 45 minutes 9. Allow to cool to room temperature 10. Add 100ul Protein Precipitation Solution 11. Vortex vigorously for 20 seconds 12. Precipitate on ice for 5 minutes 13. Pellet at 13,000-16,000g for 3 minutes 14. Transfer supernatant into clean 1.7ml tube containing 300ul 100% isoproponol 15. Mix sample by inverting 50 times 16. Centrifuge 13,000-16,000g for 1 minute to pellet DNA 17. Discard supernatant 18. Wash pellet with 300ul 70% ethanol by inverting tube several times 19. Centrifuge 13,000-16,000g 1 minute. Carefully pour off ethanol. Pellet may be loose. 20. Invert and drain tube, allow to air dry for 10-15 minutes 21. Add 50ul DNA hydration solution 22. Incubate at 65C for 5 minutes (up to an hour). Additional incubation at room temperature may be required. 23. Mix gently, pulse spin.
Label
Biotin
Label protocol
Genomic DNA was digested by sonication to sizes of 200-600bp, as visualized by agarose gel electrophoresis, then purified by using the Qiagen Qiaquick PCR purification kit. Purified fragments (1 to 2 ug) of test strain as well as reference strain 0140J were labeled with Biotin, using Mirus Label IT® µArray® Biotin Labeling Kit (Cat #MIR 8050, Mirus Corp., Madison, WI) according to the manufacturer’s instructions, followed by removal of unincorporated dyes using Qiaquick (Qiagen) columns and then hybridized to the microarray.
1. Grow overnight culture of Streptococcus uberis in Todd Hewitt Broth at 37C 2. Pipet 1ml of culture into 1.7ml microfuge tube 3. Pellet cells at 13,000-16,000g for 15 seconds. Discard supernatant 4. Add 300ul of Genomic DNA lysis Solution and pipet up and down to suspend 5. Add 1.5ul of 4,000u/ml lysozyme (up to 10,000u/ml) 6. Incubate at 80C for 5 minutes to lyse cells 7. Add 1.5ul RNase solution (4mg/ml) to lysate 8. Mix by invertin 25 times and incubate at 37C for 45 minutes 9. Allow to cool to room temperature 10. Add 100ul Protein Precipitation Solution 11. Vortex vigorously for 20 seconds 12. Precipitate on ice for 5 minutes 13. Pellet at 13,000-16,000g for 3 minutes 14. Transfer supernatant into clean 1.7ml tube containing 300ul 100% isoproponol 15. Mix sample by inverting 50 times 16. Centrifuge 13,000-16,000g for 1 minute to pellet DNA 17. Discard supernatant 18. Wash pellet with 300ul 70% ethanol by inverting tube several times 19. Centrifuge 13,000-16,000g 1 minute. Carefully pour off ethanol. Pellet may be loose. 20. Invert and drain tube, allow to air dry for 10-15 minutes 21. Add 50ul DNA hydration solution 22. Incubate at 65C for 5 minutes (up to an hour). Additional incubation at room temperature may be required. 23. Mix gently, pulse spin.
Label
Biotin
Label protocol
Genomic DNA was digested by sonication to sizes of 200-600bp, as visualized by agarose gel electrophoresis, then purified by using the Qiagen Qiaquick PCR purification kit. Purified fragments (1 to 2 ug) of test strain as well as reference strain 0140J were labeled with Biotin, using Mirus Label IT® µArray® Biotin Labeling Kit (Cat #MIR 8050, Mirus Corp., Madison, WI) according to the manufacturer’s instructions, followed by removal of unincorporated dyes using Qiaquick (Qiagen) columns and then hybridized to the microarray.
Hybridization protocol
Standard hybridization conditions for the biotinylated target included preblocking for 30 min at 50°C with 6x SSPE (1x SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH 7.7]) containing 0.05% Tween 20, 5x Denhardt's solution, and 100ng/ul salmon sperm DNA followed by hybridization of the biotinylated target in hybridization solution (6x SSPE, 20-50ng/ul labeled DNA and 0.05% SDS) overnight at 50°C. The arrays were then washed once for at least 15 min with SSPE wash 1 (6x SSPE, 0.05% Tween-20) and then for 1 min with each of the following solutions: SSPE wash 2 (3x SSPE, 0.05% Tween-20), SSPE wash 3 (0.5x SSPE, 0.05% Tween-20), and PBST wash (2x PBS, 0.1% Tween-20), followed by a final 2x PBS wash at room temperature. The hybridized array was then blocked with 5X PBS-Casein Blocking Buffer (Cat # P804901, BioFX Laboratories, Owings Mills, MD) for 15 min at room temperature and labeled for 30 min with Cy5-streptavidin (Cat # PA45001, GE Healthcare, Amersham Biosciences, Piscataway, NJ) using the 1 mg/ml stock solution diluted 1:1,000 in 5X PBS-Casein buffer. The arrays were scanned after they were washed twice with each of PBST and PBS solutions.
Scan protocol
Hybridized microarrays covered with the imaging solution and the coverslip were scanned with an Axon GenePix™ 4000B laser scanner (Axon Instruments, Union City, CA) using the following parameters:Wavelengths=635, ScanPower=100, FocusPosition=100, PixelSize=5 .
Description
test strain supplementary file names: D2S2H4.gpr (array 4) D2S3H4.gpr (array 3) reference strain supplementary file names: D2S1H1.gpr (array 3) D2S1H3.gpr (array 2) D2S2H1.gpr (array 2) D2S3H1.gpr (array 4) Array numbers of referenced GPR files (array 1, array 2, array 3, array 4) can be found in the referenced platform's full_array.txt.gz file, CombiMatrixdesign #4887
Data processing
After scanning, the image was saved as a .tiff file from which the data were extracted using GenePix software. The raw hybridization signal values, displayed in the GenePix Pro analysis Results (GPR) file, were log2-transformed to be used in subsequent analysis. Signal intensities of replicate microarrays of the reference strain 0140J were averaged, and served as denominator to obtain log-ratio values of the intensities between each array of test strain and reference strain. The log-ratio values for each array were normalized by median subtraction so that the median log ratio of each array was equal to 0. The normalized log2 (test/reference) values of replicate arrays for each test strain were averaged to determine the presence or absence of a gene in each strain. The cutoff was empirically set at -2.
