6 protocols
AccessionType
feature_extraction
Title: Affymetrix CEL analysis. Description:
nucleic_acid_extraction
The cells were then incubated with formaldehyde (final concentration, 1%) for 30 min at room temperature to crosslink Hha to the chromosomal DNA, washed with TBS (pH 7.5), and stored at -80 degrees C. The collected cells were disrupted by sonication on ice in 3 ml of UT buffer (100 mM HEPES, 50 mM imidazole, 8 M urea, 0.5 M NaCl, 1% Triton X-100, and 10 mM beta-mercaptoethanol, pH 7.4) containing a protease inhibitor cocktail (Roche, Germany). A part of supernatant fraction (whole cell extract) in each sample was recovered and proteins in whole cell extracts were digested with 2 mg/ml proteinase K (Takara, Japan) at 42 degrees C for 2 hr, followed by incubation at 65 degrees C for 6 hr to decrosslink the interaction of proteins and the chromosomal DNA, and to inactivate the proteinase K. Free DNA fragments in the whole cell extracts (sup DNA) were purified with a QIAquick purification kit (Qiagen, Germany) and eluted with 100 ul of the provided elution buffer. The recovered DNA was PCR amplified as described previously (see reference). Reference Katou, Y., Kaneshiro, K., Aburatani, H. and Shirahige, K. 2006, Genomic approach for the understanding of dynamic aspect of chromosome behavior, Methods Enzymol., 409, 389-410. (Parameters: Extracted product = genomic_DNA, Amplification = none)
nucleic_acid_extraction
The cells were then incubated with formaldehyde (final concentration, 1%) for 30 min at room temperature to crosslink Hha to the chromosomal DNA, washed with TBS (pH 7.5), and stored at ?80 degrees C. The collected cells were disrupted by sonication on ice in 3 ml of UT buffer (100 mM HEPES, 50 mM imidazole, 8 M urea, 0.5 M NaCl, 1% Triton X-100, and 10 mM beta-mercaptoethanol, pH 7.4) containing a protease inhibitor cocktail (Roche, Germany). After centrifugation at 15,000 rpm for 30 min, 25 ul of Dynabeads His-Tag Isolation & Pulldown (Invitrogen, Norway) was added to the supernatant, which was then incubated for 10 min at 4 degrees C with gentle shaking. The beads were washed five times with UT buffer, and then the bead-bound Hha complexes were eluted twice with 400 ul of elution buffer (100 mM Tris–HCl pH 7.5, 0.5M imidazole, and 1% SDS). The eluted complexes were passed through Microcon-10 (Millipore, USA), and complexes retained on the membrane were washed three times with wash buffer (50 mM Tris.HCl pH 7.5, 1% SDS, and 10 mM EDTA). His-tagged Hha in whole cell extracts and immunoprecipitated DNA fractions were digested with 2 mg/ml proteinase K (Takara, Japan) at 42 degrees C for 2 hr, followed by incubation at 65 degrees C for 6 hr to decrosslink H-NS-Flag and the chromosomal DNA, and to inactivate the proteinase K. Free DNA fragments in affinity purified DNA fractions (ChAP DNA) were purified with a QIAquick purification kit (Qiagen, Germany) and eluted with 100 ?l of the provided elution buffer. The recovered DNA was PCR amplified as described previously (see the reference added at the end of this discription). Reference Katou, Y., Kaneshiro, K., Aburatani, H. and Shirahige, K. 2006, Genomic approach for the understanding of dynamic aspect of chromosome behavior, Methods Enzymol., 409, 389-410. (Parameters: Extracted product = genomic_DNA, Amplification = none)
grow
W3110 pQE80Hha (TU08) and W3110 hns::km stpA::Cm pQE80Hha (TU10) cells were grown in 50 ml of LB (+0.3 M NaCl) medium with aeration at 37 degrees C until the culture reached an OD600 of 0.1. Then, isopropyl beta-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and the cultivation was continued to an OD600 of 0.4, for expression of N-terminally 6xHis-tagged Hha. (Parameters: time unit = seconds, min temperature = 37, temperature unit = C)
hybridization
According to the manufacturer's instruction for Prokayotic Target Hybridization by Affymetrix (Parameters: Chamber type = Affymetrix- GeneChip Hyb Oven 640, Quantity of label target used = 4, Mass unit = Micro gram, time = 16, Tiny time unit = hours, Volume = 200, Volume unit = Micro litre, temperature = 45)
labeling
PCR-amplified DNA fragments were digested by DNase I (GE healthcare Bioscience) treatment. The digested DNA fragments were terminally labeled with biotin-ddUTP using ENZO BioArray Terminal Labeling Kit (ENZO life sciences) (Parameters: Amplification = none, Mass unit = Micro gram)