8 protocols
For hybridisation on 105k arrays, 30-40 pmols of Ab-sample was mixed against 30-40 pmols of No Ab treated sample. The volume was adjusted to 260 uL with sterile nuclease-free water and 26 uL of blocking agent was added in each tube and the samples were vortexed. 130 uL of hybridisation buffer were added and the samples were pipetted 10 times avoiding bubble formation. The samples were denatured at 94C for 3min, spin briefly and added on the slides. The slides were incubated at 70C for 40h at 20rpm. For washing, each slide was disassembled into Wash 1 cGH buffer (Agilent) and transferred to a rack into another glass container full of Wash 1 cGH buffer (Agilent) and mixed for 5 min. The slides were transferred into warm Wash 2 cGH buffer (37C) (Agilent)and mixed for 1 min. Then they were transferred into Acetonitrile filled container and mixed for 1 min and the into drying and stabilisation solution (Agilent) and mixed for 30 sec.
Data normalilsed within the statistical programming environment R using the package LIMMA. Arrays were normalised using the across-array scale function only. The values in the Reporter REF column of this file map to the Comment[AEReporterName] column of the array design file.
Use of an Agilent dual laser 5 um scanner; at 532 nm for the cy3 channel and 635 nm for the cy5 channel.
Incubate at 30C in 500 ml baffled flasks.
Agilent Technologies Image Analysis and Feature Extraction software (Version 10.5)
Add 4.00E+06 spores to 160 ml of NB.
Add formaldeyde to the 20 ml culture to a final concentration of 1% and incubate at 30C in a shaking incubator for 20 min. Add glycine to 0.5M final concentration to the culture and incubate at 30C in a shaking incubator for 5 min.The bacterial pellets were placed PTFE shaking flasks previously dipped in liquid nitrogen. and the mycelium was mechanically disrupted by using a Mikrodismembrator U mechanical device (Sartorius Stedim Biotech) in PTFE shaking flasks previously dipped in liquid nitrogen. A chromium steel grinding ball and the contents of a lysing matrix B tube (Q-BIOgene) were added to the PTFE flasks containing the mycelial pellet previously frozen by immersion in liquid nitrogen; the mycelium was disrupted by shaking either once or twice for 2 min at 2,000 rpm while kept frozen at all stages by submerging into liquid nitrogen. The pulverized mycelium was transferred to a 15 mL falcon tube and 3.5 mL of IP buffer ( (50 mM Tris-HCl, pH8, 150 mM NaCl, 0.5% Triton X-100 plus protease inhibitor tablets; 1 tablet per 10 mL) were added. After mixing by pipetting the samples were sonicated on ice at 50% power x4 20 sec ON, 40 sec OFF (Sonics VibraCell VCX130). The samples were then centrifuged at 12,000 rpm for 25 min. The supernatant of each tube was frozen at -20C. In order to test if the desired range of fragments was obtained by sonication (0.5-1Kb fragments) 70 uL from each chromatin sample were incubated with 20 uL of 5x Elution buffer and 10 uL of proteinase K at 42C for 2 h and then at 65C for 6 h (a PCR machine was used). 10 uL samples were then loaded on a 1% agarose gel together with a molecular size marker. 800 uL of chromatin to be used in one immunoprecipitation reaction (either with anti-AbrC3 polyclonal antibodies or control immunoprecipitation with no antibodieas added) were pre-cleared with 50 uL of Ultralink immobilized Protein A/G sepharose beads (Pierce) by rotating for 4 h at 20rpm. 800 uL of immunoprecipitated chromatin were incubated either with 10 ug of anti-AbrC3 polyclonal antibody or with 10 uL of nuclease free water (No antibody) as control at 4C overnight, at 20rpm. The following day the samples were incubated with 80 uL of Ultralink immobilized Protein A /G sepharose beads (Pierce) for 4 h. The sepharose beads were pelletted by centrifugation at 3300 rpm for 1 min and washed with 750 uL IP buffer plus protease inhibitor cocktail (Roche) followed by 1 mL IP+salt buffer with 500 mM NaCl), 1 ml of wash buffer (wash 3, 10 mM Tris pH 8, 250 mM LiCl, 1 mM EDTA, 0.5% nonidet P-40 and 0.5% Na deoxycholate) and 1 ml of TE pH 7.6 (wash 4), with incubation at 4C in a rotating wheel for 15 min and centrifugation at 3,300 rpm for 1 min. After the first wash the protein A/G bound DNA protein complexes were transferred to a non-stick microfuge tube. (plus protease inhibitor cocktail. Finally, the beads were re-suspended with the 240 uL of Elution buffer plus 10 uL of Proteinase K (Roche) and incubated at 55C overnight. The samples were then de-cross linked at 65C for 30 min and the supernatant was transferred to a fresh tube. The beads were washed with 50 uL wit TE buffer (pH 7.6) and the supernatants were pooled in the fresh tube. 500 uL of TE buffer were added and the samples were extracted twice with phenol:chloroform:isoamyl alcohol 25:24:1 and once with chloroform (vortex 10 sec, centrifuge at 13K for 10 min, 4C). 1/10 V of sodium acetate (pH 5.5), 2.5 V of ice-cold ethanol and 20 ug of glycogen were added to the supernatant and the samples were vigorously agitated on a vortex mixer and incubated at -70C for 30 min. A 30 min spin followed at 13 K at 4C. The pellets were washed with 180 uL of 75% ethanol, air-dried and re-suspended in 25 uL of nuclease-free water. The concentration was determined by using the Nanodrop spectrophotometer.
Immunoprecipitated DNA were labelled with either Cy3-dCTP and Cy5-dCTP according the experimental design, using the BioPrime kit (Invitrogen). DNA (0.1-1 ug) was denatured at 94C for 3 min in 40 ul including 20 ul 2.5X random primer mix and kept on ice. 5 ul nucleotide mix (2 mM dATP, 2 mM dGTP, 2 mM dTTP, 0.5 mM dCTP), 3.75 ul Cy3/Cy5-dCTP (Perkin Elmer) and 1.5 ul of Klenow fragment were added and the reaction was incubated at 37C overnight. The labelled DNA was purified using the MinElute PCR purification kit (Qiagen) and the incorporated Cy3/Cy5-dCTP was quantified with the NanoDrop ND-1000 spectrophotometer.