Generating a Temperature Sensitive Allele using Hydroxylamine
Preparation for mutagenesis:
1. Generate URA3-marked and LEU2-marked plasmids containing your gene. Make
sure your construct complements the null phenotype before proceeding (transform
URA3-marked plasmid containing your gene into heterozygous null/+. Dissect
tetrads and make sure that all nulls are URA+ and 5-FOA-. Transform LEU2marked plasmid containing gene or LEU2-,marked plasmid alone into haploid
null covered by URA3-marked plasmid containing gene. Make sure that only
gene-containing LEU2-marked plasmid complements 5-FOA sensitivity).
2. Generate a haploid strain that has a null mutation in the gene of interest and
carries a URA3-marked plasmid.
Mutagenesis
3. Combine in a total volume of 600µl:
LEU2-marked plasmid DNA (100µg)
0.5M hydroxylamine hydrochloride
50mM sodium pyrophosphate pH 7.0
100mM NaCl
2mM EDTA
4. Incubate at 75ºC
5. Stop reactions by placing 100µl on ice at 0, 20, 40, 60, 80, and 100 min. time
points
6. Clean up on PCR cleanup columns. Elute in 30µl EB.
7. Transform 0.5µl into leuB- bacteria and plate on LB-Amp. Patch 100 colonies
onto M9-leucine. Select a mutagenesis rate that gives 5-10% LeuB- AmpR
bacteria.
8. Transform 10µl into 50µl competent haploid mutant/URA3 plasmid carrying
yeast. Resuspend transformation in 1ml SD-LEU + 25% glycerol and freeze in
100µl aliquots. Thaw one aliquot and plate 10, 50, and 100µl to get
#transformants/µl.
9. Plate to get 250 colonies/plate on 20 plates Grow 2 days at 25ºC. Replica plate
onto 5-FOA at 25ºC. Grow 3 days at 25ºC. Replica plate.
10. Restreak 5-FOA 37ºC-, 25ºC+ cells from 25ºC plate onto –LEU, 5-FOA and retest
temperature sensitivity.
11. Rescue plasmid and transform heterozygous null. Dissect tetrads and confirm
temperature sensitivity.