Team:HKUST/Protocols/Transformation to Ecoli

Transformation to yeast

Purpose: To transform desired constructed plamid DNA to yeast to test its effect.

Materials: LiOAc solution, PEG4000 solution, DMSO (dimethyl sulfoxide), yeast strain, desired plasmid, YEPD, SD media with specific selection marker absent (-Leu, -His, or -Ura), ddH2O

Procedure:

a.A stationary culture of yeast grown in YEPD is used to inoculate 10mL of YEPD in a 100mL Pyrex flask.
b.Cells are grown at 30 °C with shaking (200 rpm) until a density of 1-4x107 is reached (OD660=0.4).
c.Yeast is transformed into 4 sterile 1.5mL tubes and are centrifuged for 2mins (4,000 rpm).
d.Damp off media and wash the pellet with 100μL ddH2O (to culture 1:1). Resuspend the cells by gently shake it or flip it.
e.Centrifuge it again for 2mins (4,000 rpm). Remove the supernatant.
f.Wash the pellet with 1mL LiOAc solution and pour it into a single tube. Resuspend the cell by gently shake it or flip it.
g.Repeat step e and f once.
h.Add 100μL of yeast suspension to a 1.5mL microcentrifuge tube, and add 10μL of DNA to be transformed. Mix them gently and stand the tube at room temperature for 5mins.
i.Add 280μL PEG4000 solution. Mix it gently by inverting 4-6 times, then the tube is stored at 30 °C for 45mins without shaking.
j.Add 43μL DMSO to give an approximate 10% (volume percentage) DMSO solution. Mix the solution gently by inverting.
k.Heat shock at 42°C for 5mins.
l.Centrifuge it for time long enough to get pellet (usually 5 sec) at 12,000rpm.
m.Remove the supernatant and wash it with ddH2O.
n.Centrifuge it again for 50sec (13,000 rpm). Remove the supernatant.
o.Resuspend the cell with 1mL ddH2O.
p.Plate the solution on SD media with specific selection marker absent.

Tips:

Ignite the fire for sterile environment when transforming the yeast cell, since there is no any antibiotic in the culture.br>

Safety tips:

Be careful with the fire, and extinguish it after use.

• 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