Team:HKUST/Protocols/Yeast genomic DNA extraction

Yeast genomic DNA extraction

Purpose: To extract yeast genomic DNA from yeast strain (strain YPH501, in our case).

Materials: yeast strain, lysis buffer, phenol, chloroform, 3M NaAc, 100% ethanol, 70% ethanol, ddH2O, glass beads.

Procedure:

a.Add 200μL lysis buffer.
b.Pick a whole patch of colony and add it in the tube.
c.Add 100μL phenol, and mix it well.
d.Add 100μL chloroform, and a few glass beads.
e.Vortex for 4-5mins and centrifuge for 5mins (13,000 rpm).
f.Put the supernatant (~160μL) to another tube.
g.Add 200μL phenol/chloroform (1:1 in volume)
h.Vortex for 4-5mins and centrifuge for 5mins (13,000 rpm).
i.Put the supernatant (~120μL) to another tube.
j.Add 0.1 volume NaAc(pH5.2), approximately 12μL.
k.Add 2 volume of 100% ethanol, approximately 240μL.
l.Store it at -20 °C for 20mins.
m.Centrifuge for 10mins (13,000 rpm) and remove the supernatant.
n.Add 2 volume of 70% ethanol.
o.Centrifuge for 5mins (13,000 rpm) and remove the supernatant.
p.Stand the tube without closing it for a while to let the ethanol evaporate.
q.Add 20μL ddH2O to resuspend it.
r.Store it at -20 °C.

Safety tips:

Phenol is a strong toxin, make sure to wear gloves and be careful with this chemical.

• Agarose gel preparation and gel electrophoresis
• PCR (Taq, Vent)
• PCR product clean-up
• Enzyme digestion (vector, insert)
• Plasmid DNA extraction
• Gel cutting
• Gel extraction
• Ethanol precipitation
• Ligation
• Transformation to E.coli
• Transformation to yeast
• Cell lysis
• Yeast genomic DNA extraction
• Western blotting
• GFP testing