|
| Status |
Public on Dec 05, 2008 |
| Title |
WT(-van)vssigM(-van)_rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
WT/-van
|
| Organism |
Bacillus subtilis |
| Characteristics |
WT/-van
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cell cultures were grown to an OD600 of 0.4 and split into 2 flasks with equal volume, vancomycin was added to 1 flask to a final concentration of 2 microgram/ml and cells were harvested 10 min after treatment. RNA isolation was performed using the RNeasy mini kit (Qiagen). RNA was subsequently DNase treated with TURBO DNA-freeTM (Ambion) and precipitated overnight. The RNA was dissolved in RNAse free water and quantified using a NanoDrop spectrophotometer (Nanodrop Tech. Inc., Wilmington, DE).
|
| Label |
Alexa Fluor 555
|
| Label protocol |
cDNA synthesis was performed using the SuperScriptTM Plus Indirect cDNA labeling System (Invitrogen) as per the manufacturer’s instructions using 20 microgram of total RNA. Total cDNA was labeled overnight with Alexa Fluor 555 or Alexa Fluor 647 (Invitrogen)
|
| |
|
| Channel 2 |
| Source name |
sigM/-van
|
| Organism |
Bacillus subtilis |
| Characteristics |
sigM/-van
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cell cultures were grown to an OD600 of 0.4 and split into 2 flasks with equal volume, vancomycin was added to 1 flask to a final concentration of 2 microgram/ml and cells were harvested 10 min after treatment. RNA isolation was performed using the RNeasy mini kit (Qiagen). RNA was subsequently DNase treated with TURBO DNA-freeTM (Ambion) and precipitated overnight. The RNA was dissolved in RNAse free water and quantified using a NanoDrop spectrophotometer (Nanodrop Tech. Inc., Wilmington, DE).
|
| Label |
alexa Fluor 647
|
| Label protocol |
cDNA synthesis was performed using the SuperScriptTM Plus Indirect cDNA labeling System (Invitrogen) as per the manufacturer’s instructions using 20 microgram of total RNA. Total cDNA was labeled overnight with Alexa Fluor 555 or Alexa Fluor 647 (Invitrogen)
|
| |
|
| |
| Hybridization protocol |
Equal amounts (100-150 pmol) of labeled cDNA were combined plus hybridization buffer (2X = 50% formamide, 10X SSC, 0.1% SDS). cDNA mix was denatured at 95oC and hybridized 16-18 hours at 42oC to DNA microarray slides which had been prehybridized for at least 30 min at 42oC in 1% bovine serum albumin, 5X SSC (1X SSC is 0.15 M NaCl and 0.015 M sodium citrate), 0.1% sodium dodecyl sulfate (SDS), washed in water and dried. Following hybridization the slides were washed sequentially in: 2X SSC + 0.1% SDS for 5 min at 42oC, 2X SSC + 0.1% SDS for 5 min at room temperature, 2X SSC for 5 min at room temperature, 0.2X SSC for 5 min at room temperature, and finally dipped in water and spun until dry.
|
| Scan protocol |
Arrays were scanned using a GenePixTM 4000B array scanner (Axon Instruments, Inc.)
Raw data files were produced from the scanned images using the GenePix Pro 4.0 software package (GPR files).
|
| Description |
Alexa Fluor 555 (WT/-van) / 647 (sigM/-van)
|
| Data processing |
Red/green fluorescence intensity values were normalized using the GenePix Pro 4.0 software package such that the ratio of medians of all features was equal to 1
|
| |
|
| Submission date |
Dec 04, 2008 |
| Last update date |
Feb 20, 2014 |
| Contact name |
John D. Helmann |
| E-mail(s) |
[email protected]
|
| Phone |
607 255 6570
|
| Organization name |
Cornell University
|
| Department |
Microbiology
|
| Street address |
372 Wing Hall
|
| City |
Ithaca |
| State/province |
NY |
| ZIP/Postal code |
14853 |
| Country |
USA |
| |
|
| Platform ID |
GPL7420 |
| Series (1) |
| GSE13820 |
Sigma(M) regulated genes under antibiotic stress (vancomycin) |
|
