Tuesday, November 18, 2003 Description In vitro Mutagenesis with dut ung single stranded DNA Procedure In an 0.5 ml microfuge tube add: 0.2 microgram single stranded dut ung DNA 1.3 microliter kinased oligo from above (33 ng) 1 microliter...
Tuesday, November 18, 2003 Description Replication timing using transient hemimethylation Procedure 1. Grow ~330 ml of RM14-3a expressing dam methylase (RM14-3a Meth5) to an OD660 of about 0.3 (3.85 x 106 cells/ml) at 23oC. Arrest with 200...
Tuesday, November 18, 2003 Description Replication timing by density transfer Procedure 1. Grow cells at least 7 generations in 0.1% (w/v) [13C]-acetate or glucose 0.01% (w/v) [15N]-ammonium sulfate Amino acids/supplements (as required, ad...
Tuesday, November 18, 2003 Description Replication timing by comparative hybridization Procedure 1. Grow the cells in minimal medium with 1% Na-acetate or 3% glycerol as the carbon source, aiming for about 20 ml cells per time point. (Raff...
Thursday, November 13, 2003 Description Method for assaying gliotoxin production in Aspergillus fumigatus Procedure Culture and extraction Culture Aspergillus fumigatus on Sabouraud agar plates for 2 days at 37C and harvest the conidia usi...
Thursday, November 13, 2003 Description A rapid method for generating gene deletions in Aspergillus fumigatus Procedure Step 1: Introduction of target cosmid into KS272(pKOBEG) The E. coli strain KS272(pKOBEG) is transformed with the relev...
Tuesday, October 21, 2003 Description Random Primed Labeling of DNA Procedure 1. Denature 30 to 50 ng of DNA by boiling for 2 min. 2. To a microcentrifuge tube add in this order: ddH2O to give a final volume of 50 ul 10 ul of OLB Denatured...
Tuesday, October 21, 2003 Description Labeling Oligonucleotides Using T4 Polynucleotide Kinase Procedure 1. Add the following to a microcentrifuge tube: 2 l of 10X Kinase Buffer 3 l of -[32P]-ATP (7000 Ci/mmole or 167 Ci/ul) (CAUTION! See...
Description S1 Nuclease Mapping Using Single-Stranded Radiolabeled Oligonucleotide Probes Procedure 1. Mix in a sterile microcentrifuge tube: 10 ìl of End-labeled, single-stranded, radiolabeled oligonucleotide probe (dilute so that there...
Sunday, October 19, 2003 Description An end labeled DNA probe is incubated with a purified DNA-binding factor or with a protein extract. The unprotected DNA is then digested with DNase I such that on average, every DNA molecule is cut once...