HIS3, HIS4, Ty1 plasmids also with URA3 and GAL1 promoter
Extracted molecule
other
Extraction protocol
standard yeast plasmid DNA preparation
Label
Cy5
Label protocol
Each plasmid was digested in three parallel reactions with AluI, MspI, and HpyCH4V. The resulting fragments were heat-inactivated, pooled and labeled for hybridization to the microarray as follows: 200 ng DNA was incubated with 36 micrograms random hexamer in a 23 microliter reaction at 100C for 2 minutes, then 4C for 4 minutes. The labeling reaction then proceeded with the addition of 5 microliters 10x dNTP (8 mM dATP, dCTP, dGTP, 4 mM dUTP), 5 microliters 10x Klenow buffer, 7 microliters Klenow (exo-) fragment (5U/microliter/), 7 microliters H2O, and 2 microliters Cy5 dUTP, and was incubated at 37C for 2 hours. The reaction was stopped with 5 microliters 0.5 M EDTA pH 8.0. The products were mixed with 450 microliters TE and concentrated on a Microcon YM-30 (Amicon catalog #42410) column. The products were washed again with 450 microliters TE and 10 microliters sheared salmon sperm DNA (10 mg/ml), and concentrated again on a Microcon column. The resulting volume was adjusted to 26 microliters with the addition of H2O, and SDS and SSC were added to final concentrations of 3x SSC and 0.3% SDS, in a total volume of 32.5 microliters. After incubation at 100C for 90 seconds and then 37C for 30 minutes, the products were spotted onto microarrays and covered with VWR 22x60mm cover slips.
Description
See above.
Data processing
the value given is simply the log2 of the difference between F635 and B635
