4 protocols
Array Hybridization 1) Preheat hybridization oven to 45 degrees C. 2) Fill each S. cerevisiae genome tiling array chip with 1x hybridization buffer and incubate for at least 10 minutes in the hybridization oven at 45 degrees C, spinning at 60 rpm. 3) Prepare chip hybridization master mix. For each chip add 150 ul 2x hybridization buffer, 94 ul ddH2O, 7.5 ul 20 mg/ml BSA, 5.6 ul 50 nM b213 control oligonucleotide and 3 ul 10 mg/ml herring testes carrier DNA. 4) Add the labeled DNA sample (45 ul) to 255 ul of chip hybridization master mix. 5) Heat the hybridization mix at 95 to 100 degrees C for 10 minutes. 6) Cool on ice for 5 minutes. 7) Remove 1x hybridization buffer from each chip. 8) Fill each chip with the DNA hybridization mix (see Note 7). Cover each gasket with a tough-spot to prevent leakage. 9) Place chips in oven and hybridize for 20 hours at 45 degrees C, spinning at 60 rpm. 10) After hybridization remove the hybridization solution and save in its corresponding tube at 4 degrees C. 11) Fill each chip with 1x hybridization buffer.
(Parameters: Chamber type = Affymetrix- GeneChip Hyb Oven 640, Quantity of label target used = 15, Mass unit = Micro gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 45)
Extract genomic DNA: 1) Extract genomic DNA using YeaStar Genomic DNA kit (cat # D2002) Protocol I or with equivalent product with the following modifications: a) Spin 5 x 10^7 cells at 2700 x g for 2 minutes. b) Incubate with R-zymolyase (provided in kit) for 60 min and vortex for 1 minute. c) Vortex YD lysis buffer with sample for 1 min at medium-low setting. d) Centrifuge times all solutions through columns at 17900 x g for 2 minutes. e) Elute in 60 ul of 10 mM Tris-HCl and centrifuge for 30 seconds. 2) Measure concentration of genomic DNA. 3) Concentrate genomic DNA if concentration is less than 5 ng/ul. Whole Genome Amplification 1) Amplify genomic DNA prepared with genomic DNA mini-prep using GenomePlex complete whole genome amplification kit (Sigma cat # WGA2) with the following modifications: a) Prepare a 5 ng/ul DNA solution from extracted DNA. b) Add 10 ul of 5 ng/ul DNA solution to 1 ul of 10x fragmentation buffer. 2) Measure amplified DNA concentration. 3) Purify final product before labelling.
(Parameters: Extracted product = genomic_DNA, Amplification = PCR)
DNA Fragmentation 1) Prepare DNase master mix: 7.4 ul ddH2O, 0.2 ul 10x one-phor-all buffer, 1.2 ul 30 mM CoCl2 and 2 ul 1U/ul DNase I amplification grade. Mix solution with a pipette. 2) Prepare 0.403 ug/ul DNA solution with ddH2O to a total volume of 37.25 ul in a thin-walled PCR tube. Mix solution with a pipette. 3) Add to each DNA sample: 4.5 ul 10x one-phor-all buffer, 2.25 ul 30 mM CoCl2 and 1.5 ul of the DNase master mix. Mix solution with a pipette. 4) Use a thermocycler to incubate for 4 minutes at 37 degrees C, then 95 degrees C for 10 minutes, then decrease temperature to 4 degrees C. 5) Check digestion on a thin (< 5 mm) 0.1x lithium boric (LB) acid/ 2% agarose gel. Immerse gel in 0.1x LB running buffer such that there is only a thin (~1 mm) layer covering the gel. 6) Load gel with 0.5 ul (500 ng) of 10 bp ladder and 1 ul of each DNA sample plus 1 ul 5x LB loading medium and 3 ul ddH2O and run at 250V for 27 minutes. 7) Stain for 20 min in 1x SYBR green in 1x TAE. 8) Visualize with UV light. Smear of fragmented DNA should appear centered at approximately 25 bp. 9) If the smear is centered at a larger size, repeat steps 4 to 8 after adding an additional 1.5 ul of the DNase master mix to each sample. 10) Once desired fragment size is obtained, store at -20 degrees C until required for labeling. DNA Labeling 1) Add 1 ul biotin-N6-ddATP (1 nmol/ul) and 1.54 ul TdT (20U/ul) to the fragmented DNA sample. 2) Incubate for 1 hour at 37 degrees C and cool to 4 degrees C. 3) Store at 4 degrees C or use immediately. Do not freeze after labeling.
(Parameters: Amplification = none, Mass unit = Micro gram)
Yeast strains were grown at 30 degrees C in YPD to an optical density at 600nm greater than 10. Cells were collected, washed with 1X TBS buffer, frozen in liquid nitrogen, and stored at -80 degrees C overnight.
(Parameters: start time = 1, time unit = days, min temperature = 30, temperature unit = C, media = YPD)