PEG is a high-molecular polymer. During competent transformation of yeast cells, PEG with a molecular weight of approximately 3000 yields the optimal promoting effect on transformation efficiency.In yeast transformation, PEG protects the cell membrane under high-concentration lithium acetate (LiAc) conditions and alleviates structural damage to the cell membrane caused by LiAc. Meanwhile, PEG facilitates tighter adhesion of plasmids to the yeast cell wall.LiAc induces a transient competent state in yeast cells, enabling the cells to take up exogenous DNA.PEG/LiAc Solution is an essential supporting reagent for transformation of prepared yeast competent cells, and 500 µL of PEG/LiAc Solution is required for each single transformation reaction.
Product Composition and Formula:
| Product Component | Formula (ml/L) | | 50% PEG3350 | 800 | | 1M LiAc (10X) | 100 | | 10X TE Buffer | 100 |
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Operation Protocol
1. Pretreatment of Carrier DNA: Incubate Carrier DNA at 95 ℃ in a metal bath for 5 min, or in a water bath with a floating rack at 95 ℃ for 3 min, then immediately place it on ice for rapid cooling.
2. Take 100 µL yeast competent cells thawed on ice, sequentially add pre-cooled target plasmid (2–5 µg), 10 µL pretreated Carrier DNA and 500 µL PEG/LiAc Solution. Gently pipette up and down several times to mix thoroughly. Incubate the mixture in a 30 ℃ water bath for 30 min; invert the tube 6–8 times to mix at the 15 min mark.
3. Transfer the tube to a 42 ℃ water bath and incubate for 15 min; invert the tube 6–8 times to mix at the 7.5 min mark.
4. Centrifuge at 5000 rpm for 40 s and discard the supernatant. Resuspend the pellet with 400 µL ddH₂O, then centrifuge for 30 s and remove the supernatant.
5. Resuspend the cell pellet with 50 µL ddH₂O or 0.9% NaCl solution, then spread the suspension onto agar plates.
Precautions:
1. PEG tends to precipitate after long-term static storage. Be sure to shake the reagent thoroughly before each use to homogenize all components.
2. Unopened PEG/LiAc Solution can be stored long-term at 4 ℃. After opening, it is recommended to store at -20 ℃. Discard the reagent immediately once microbial contamination is observed.
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