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6. PEG/LiAc mix: 1.5 ml/15 ml 1 M lithium acetate solution,
1.5 ml/15 ml 10× TE, 12 ml/15 ml 50 % PEG4000, sterilize
by fi ltration.
7. Dimethyl sulfoxide (DMSO). No need to sterilize.
8. 0.9 % NaCl, autoclaved.
9. Sterile pipette tips, Eppendorf vials, centrifuge tubes,
Erlenmeyer fl asks, and materials and media for pre- and post-
transformation yeast cultivation (
see
Subheading
2.3
).
2.5 Plasmid Rescue
1. GTE buffer: 50 mM glucose, 25 mM Tris-Cl, 10 mM EDTA,
pH 8.0.
2. Acid-washed glass beads approx. 500-600
μ
m in diameter
(e.g., Sigma G-8772).
3. NaOH/SDS: 200 mM NaOH, 1 % SDS. Do not sterilize.
4. KAc/HAc: 3 M potassium acetate, 2 M acetic acid.
5. Isopropanol. No need to sterilize.
6. 75 % Ethanol. No need to sterilize.
7. 96 % Ethanol. No need to sterilize.
8. Sterile water.
9. Sterile pipette tips, Eppendorf vials, centrifuge tubes,
Erlenmeyer fl asks, and materials and media for yeast cultiva-
tion (
see
Subheading
2.3
).
10. Competent
E. coli
cells and materials for
E. coli
transformation
(
see
Note 9
).
3
Methods
Follow local biohazard rules and regulations when working with
transgenic yeasts. Laboratory strains of
S. cerevisiae
are usually con-
sidered low risk, but some paperwork may be needed if introduc-
ing yeast into the lab the fi rst time.
3.1 Yeast Strain
Handling and
Maintenance
Handle yeast using standard microbiology techniques, similar to
those well established for
E. coli.
1. For long-term storage, grow a fresh agar culture from a reli-
able source (
see
Note 10
). Use YEPD plates for plasmid-free
strains and selective media for strains containing plasmids.
Scrape off a couple of colonies using the blunt end of a tooth-
pick (resulting in a mix of several single-colony isolates), resus-
pend in 1 ml of 15 % glycerol in a screw-top vial, and store in
−80 °C. Such stocks last for several years, but renew them
from a fresh verifi ed culture once you notice falling viability.
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