|
| Status |
Public on Nov 29, 2010 |
| Title |
C. bescii versus C. saccharolyticus |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
C. bescii
|
| Organism |
Caldicellulosiruptor bescii |
| Characteristics |
strain: DSM 6725
|
| Growth protocol |
All Caldicellulosiruptor strains were initiated from axenic freeze-dried samples, except C. bescii, in their recommended growth medium per the DSMZ website at 70C. After the Caldicellulosiruptor species were revived, cultures were subcultured three times onto DSMZ640 medium plus cellobiose. 500 mL of batch grown culture was harvested after overnight growth for genomicDNA harvesting.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were harvested by centrifugation at 5,000 rpm and DNA was isolated, as described by Geslin et al. (J Bacteriol 185:3888), with the addition of lysozyme during cell lysis.
|
| Label |
Cy3,Cy5
|
| Label protocol |
Genomic DNA was partially digested with Hae III (NEB) and amino allyl-dUTP plus dNTPs were incorporated using Klenow fragment (NEB) and random nonamers (Sigma-Aldrich). Cy-NHS ester dyes (GE Healthcare) were incubated with aa-dUTP labeled DNA fragments to label genomic DNA fragments. Unincorporated dye was removed using Qiaquick spin columns according to manufacturer’s recommendations (Qiagen).
|
| |
|
| Channel 2 |
| Source name |
C. saccharolyticus
|
| Organism |
Caldicellulosiruptor saccharolyticus |
| Characteristics |
strain: DSM 8903
|
| Growth protocol |
All Caldicellulosiruptor strains were initiated from axenic freeze-dried samples, except C. bescii, in their recommended growth medium per the DSMZ website at 70C. After the Caldicellulosiruptor species were revived, cultures were subcultured three times onto DSMZ640 medium plus cellobiose. 500 mL of batch grown culture was harvested after overnight growth for genomicDNA harvesting.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were harvested by centrifugation at 5,000 rpm and DNA was isolated, as described by Geslin et al. (J Bacteriol 185:3888), with the addition of lysozyme during cell lysis.
|
| Label |
Cy5,Cy3
|
| Label protocol |
Genomic DNA was partially digested with Hae III (NEB) and amino allyl-dUTP plus dNTPs were incorporated using Klenow fragment (NEB) and random nonamers (Sigma-Aldrich). Cy-NHS ester dyes (GE Healthcare) were incubated with aa-dUTP labeled DNA fragments to label genomic DNA fragments. Unincorporated dye was removed using Qiaquick spin columns according to manufacturer’s recommendations (Qiagen).
|
| |
|
| |
| Hybridization protocol |
Sample preparation steps for microarray hybridization: Prepare Prehyb buffer: 225mL water, 75mL 20X SSC, 0.3g SDS, 0.3g BSA. Preheat Prehyb buffer 45 min 42 degree C. Prehybidize slides at 42 degree C for 45 min. Wash slides by dipping 5 time in sterile dH2O at RT. Dip twice in isopropanol at RT. Dry slide in slide spinner. Add 1 ul COT1-DNA (20mg/ml) to samples. Heat probe mixture to 95oC in heat block for 2 min to denature DNA, do a quick spin. Add 30 ul of 2x hyb. buffer that has been preheated to 42oC to each tube. Hybridization buffer: 27.2uL water, 20uL 20X SSC, 8uL 50X Denhardts solution, 24uL formamide, 0.8uL 10% SDS. Apply hyb. mix to slide and cover with a 20x60 mm polyethylene hydrophobic coverslip. Place slide in water proof container, cover well with foil and place in 42 degree C bath for 18-20 hours. The following is a standard hybridization method that seems to work well for this procedure: 1 wash cycle at 42 degree C with 2X SSC w/ 0.2% SDS. 1 wash cycle at RT with 0.5X SSC w/ 0.2% SDS. 3 wash cycles at RT with 0.5X SSC
|
| Scan protocol |
Images were aquired with a scanarray 4000 (PE) with 2 lasers. Channels were balanced using the line scan feature and by balancing the overall signals without the use of external standards
|
| Data processing |
Signals for each channel were obtained using ScanArray express and were background substrated using the median local intensity around the spot. Once all the slides were quantitated, data from the dye-flip was analyzed with JMP Genomics 4.0 (SAS, NC), using a mixed effects ANOVA model [Wolfinger et al. J Comput Biol 8 625-637 (2001).
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| |
|
| Submission date |
Aug 13, 2010 |
| Last update date |
Nov 29, 2010 |
| Contact name |
Robert M Kelly |
| Organization name |
North Carolina State University
|
| Department |
Chemical and Biomolecular Engineering
|
| Street address |
Campus Box 7905
|
| City |
Raleigh |
| State/province |
MI |
| ZIP/Postal code |
27695 |
| Country |
USA |
| |
|
| Platform ID |
GPL6681 |
| Series (1) |
| GSE23606 |
Phylogenetic, microbiological and glycoside hydrolase diversities within the extremely thermophilic, plant biomass-degrading genus Caldicellulosiruptor |
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