|
| Status |
Public on Jan 01, 2014 |
| Title |
Comparision of the retentostat 2 on day 44 over the retentostat 1 on day 44 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
2.44
|
| Organism |
Lactococcus lactis |
| Characteristics |
strain: KF147
sample: 2
time: day 44
|
| Treatment protocol |
Quenching Buffer 66.7 mM HEPES (pH 6.5) 60%Methanol Extraction Mixture (Prepared in a screw-cap tube) -500 µl phenol/Chloroform -30 µl10% SDS -30 µl 3 M NaAc (pH 5.2) -500 mg glass-beads (75-150 µm) -400 µl TE buffer 1.For each sample prepare a tube with Extraction Mixture
|
| Growth protocol |
Lactococcus lactis subsp. lactis strain KF147 were inoculated in 50 ml M17 broth (Terzaghi and Sandine, 1975) complemented with 0.5% glucose (w/v) and grown overnight at 30°C. Overnight cultures were harvested by centrifugation (6,000 g, 10 min., 4°C) and washed twice with physiological salt solution (0.9% NaCl in water). Next, the culture was inoculated into CDM containing 25 mM of glucose (Goffin, et al., 2010). Two independent, carbon source-limited retentostat cultivations with 1.5-l working volume were performed under anaerobic conditions, initiating from chemostat cultivation at dilution rates of 0.025 h-1 as previously described (Ercan, et al., 2013).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cultures were lysed by beat-beating and total RNA was isolated by subsequent phenol-chloroform extraction, followed by RNA purification and mRNA enrichment
|
| Label |
Cy5
|
| Label protocol |
Total RNA and enriched mRNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 3 h in the presence of 400 U SuperScript TMIII reverse transcrptase enzyme (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy3-label
|
| |
|
| Channel 2 |
| Source name |
1.44
|
| Organism |
Lactococcus lactis |
| Characteristics |
strain: KF147
sample: 1
time: day 44
|
| Treatment protocol |
Quenching Buffer 66.7 mM HEPES (pH 6.5) 60%Methanol Extraction Mixture (Prepared in a screw-cap tube) -500 µl phenol/Chloroform -30 µl10% SDS -30 µl 3 M NaAc (pH 5.2) -500 mg glass-beads (75-150 µm) -400 µl TE buffer 1.For each sample prepare a tube with Extraction Mixture
|
| Growth protocol |
Lactococcus lactis subsp. lactis strain KF147 were inoculated in 50 ml M17 broth (Terzaghi and Sandine, 1975) complemented with 0.5% glucose (w/v) and grown overnight at 30°C. Overnight cultures were harvested by centrifugation (6,000 g, 10 min., 4°C) and washed twice with physiological salt solution (0.9% NaCl in water). Next, the culture was inoculated into CDM containing 25 mM of glucose (Goffin, et al., 2010). Two independent, carbon source-limited retentostat cultivations with 1.5-l working volume were performed under anaerobic conditions, initiating from chemostat cultivation at dilution rates of 0.025 h-1 as previously described (Ercan, et al., 2013).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cultures were lysed by beat-beating and total RNA was isolated by subsequent phenol-chloroform extraction, followed by RNA purification and mRNA enrichment
|
| Label |
Cy3
|
| Label protocol |
Total RNA and enriched mRNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 3 h in the presence of 400 U SuperScript TMIII reverse transcrptase enzyme (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy3-label
|
| |
|
| |
| Hybridization protocol |
Hybridization buffer (Agilent In Situ Hybridization Kit Plus) was added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers at 65 degrees Celcius for 17 h. After hybridization, slides were washed sequential
|
| Scan protocol |
Scanned on an Agilent G2565AA scanner. Images were quantified using Agilent Feature Extraction Software (version A.7.5)
|
| Data processing |
Scanned slides were Lowess normalised using Prep (van Hijum, Applied Bioinformatics 2003 2(4):241-244. Interslide scaling was performed using Postprep (van Hijum, Applied Bioinformatics 2003 2(4):241-244).
|
| |
|
| Submission date |
Oct 21, 2013 |
| Last update date |
Jan 01, 2014 |
| Contact name |
Michiel Wels |
| E-mail(s) |
[email protected]
|
| Organization name |
NIZO food research
|
| Street address |
Kernhemseweg 2
|
| City |
Ede |
| ZIP/Postal code |
6718 ZB |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL17806 |
| Series (1) |
| GSE51494 |
Molecular and physiological adaptations of Lactococcus lactis at near-zero growth rates |
|
