Series
Kit : QuickGene DNA tissue kit S Model : QuickGene-810 Summary
Enables easy and rapid high-yield isolation of DNA from yeast (methylotrophic yeast Ogataea minuta)

Protocol

2 ml micro tube
(Harvest whole yeast cells by centrifuging after
Freeze the pellet rapidly (Immerse the tube in dry ice-ethanol for more than 10 min.)

Add yeast cell pellet

shaking culture in 5 ml YPAD at 30 C for 16 hours [OD600=app.3]) stainless-steel beads : 2 beads

Add 4.8 mm

Disrupt the pellet with MS-100R (TOMY SEIKO Co.Ltd.) (3,000 rpm, 30 sec.) Refreeze for 3 min. Disrupt with MS-100R (TOMY SEIKO Co.Ltd.) (3,000 rpm, 30 sec.)
Add MDT : 180 l and EDT : 20 l
Incubate at 55 C for 30 - 60 min. Centrifuge (15,000 rpm, room temperature, 10 sec.) Transfer the supernatant (200 l) to a new 1.5 ml micro tube

Add LDT : 180 l

Mix by vortexing for 15 sec.(maximum rotation speed) Flash spin down (several seconds) Incubate at 70 C for 10 min. Flash spin down (several seconds)
Add >99% ethanol : 240 l
Mix by vortexing for 15 sec.(maximum rotation speed) Flash spin down (several seconds) Lysate Transfer lysate into the cartridge of automatic nucleic-acid isolation system QuickGene. (If any aggregates are present in the lysate, transfer them altogether into the cartridge.) Select DNA TISSUE mode (Change elution volume to 50 l.) Press START Button Genomic DNA (elution volume : 50 l )
Perform isolation within 30 min. after lysate preparation.
Results : Isolation of genomic DNA from yeast
Genomic DNA was isolated from methylotrophic yeast Ogataea minuta using QuickGene system (QuickGene-810 and QuickGene DNA tissue kit S).
Kind of yeast : methylotrophic yeast Ogataea minuta Initial sample amount : 5 ml YPAD Shake culture at 30 C for 16 hours (OD600=app.3) Harvest whole yeast cells by centrifuging genomic DNA (elution volume : 50 l) RNase treatment : genomic DNA : 1 l RNase (10 mg/ml ) : 0.1 l (final conc. 100 ng/ l) up to 10 l with s.d.w. 37 C, 15 min.
1) Agarose Gel Electrophoresis of isolated genomic DNA
without RNase treatment M with RNase treatment
7 Lane M : -DNA/Hind III digest Lane 1 : Parental strain Lane 2 ~ 7 : Candidate null mutants
2) The yield and purity of genomic DNA (without RNase treatment)
Yield (g) 7 79.4 111.1 127.8 35.0 30.2 53.3 10.7 Purity (A260/280) 2.12 2.13 2.12 2.01 1.85 1.99 1.67
The use of QuickGene system enables the high-yield isolation of high-purity genomic DNA from yeast.
(Contributed by Glycobiosynthesis Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology)
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