I want to create plasmid through budding yeast in-vivo gap repair mechanism. My plan is to linearize my plasmid via PCR, and amplify my target gene with 50 bp homology to either end of the linearized

Question

Accepted Answer

In budding yeast (*Saccharomyces cerevisiae*), in vivo gap repair—also known as gap-repair cloning (GRC) or DNA assembly—is a highly efficient method for constructing plasmids by exploiting the organism's robust homologous recombination (HR) machinery (Direct, High; PMID: 33138080). This process allows multiple DNA fragments with overlapping terminal sequences to be accurately fused in a single step (Direct, High; PMID: 19074487, PMID: 26330622).

## DNA Preparation and Design
The success of gap repair depends on the length of the homology arms and the quality of the PCR fragments.

*   **Homology Arm Design:** For efficient assembly of 3–5 gene pathways (~10 kb), homology overlaps of approximately 50 bp typically yield efficiencies of 80–100% (Direct, High; PMID: 19074487). While overlaps as short as 30 bp can be used, longer overlaps (e.g., 40–50 bp) are generally recommended to ensure high accuracy and colony yields (Direct, High; PMID: 9016579).
*   **Vector Linearization:** The vector backbone can be linearized either by restriction enzyme digestion at a unique site or via PCR (Direct, High; PMID: 26330622, PMID: 20300182).
*   **Insert Amplification:** Target genes are amplified using high-fidelity DNA polymerase (e.g., Phusion) (Direct, High; PMID: 19074487, PMID: 26330622). Primers must be designed such that the 5' ends of the forward and reverse primers contain the sequence homologous to the adjacent fragment (either the linearized vector or another insert) (Direct, High; PMID: 19074487).
*   **DNA Concentration:** It is recommended to use approximately 100 ng of each DNA fragment per transformation (Direct, High; PMID: 26330622). For complex pathways, increasing the molar ratio of inserts to the vector backbone can further improve assembly efficiency (Direct, High; PMID: 19074487).

## High-Efficiency Transformation Protocol
The most common method for introducing DNA into budding yeast is the Lithium Acetate (LiAc)/single-stranded carrier DNA (ssDNA)/Polyethylene Glycol (PEG) protocol (Direct, High; PMID: 35781197, PMID: 26330622).

1.  **Preparation of Competent Cells:**
    *   Inoculate yeast into YPDA or YEPD broth and grow overnight at 30°C until the culture reaches logarithmic or early stationary phase (Direct, High; PMID: 35781197, PMID: 26330622).
    *   Pellet approximately 1–3 × 10⁸ cells (1.5 mL of culture) by centrifugation at 2500–3000 x g for 15 seconds and discard the supernatant (Direct, High; PMID: 35781197).
    *   Wash the cell pellet once with 0.1 M LiAc (Direct, High; PMID: 26330622).
2.  **Transformation Mix:**
    *   Add 5 µL of boiled, ice-chilled ssDNA (10 mg/mL carrier DNA) to the cell pellet (Direct, High; PMID: 35781197).
    *   Add the DNA fragments (linearized vector and inserts). The total volume of DNA should ideally be ≤20 µL to prevent a decrease in efficiency (Direct, High; PMID: 26330622).
    *   Add 500 µL of PLTE mix (consisting of 40% PEG-3350, 0.1 M LiAc, 10 mM Tris-HCl, and 1 mM EDTA) (Direct, High; PMID: 35781197).
    *   Add 50–56 µL of DMSO (approximately 10% final concentration) and vortex briefly to resuspend the cells (Direct, High; PMID: 35781197).
3.  **Heat Shock and Recovery:**
    *   Incubate the mixture at 30°C for 15–30 minutes (Direct, High; PMID: 35781197, PMID: 26330622).
    *   Perform heat shock in a 42°C water bath for 15–20 minutes (Direct, High; PMID: 35781197, PMID: 26330622).
    *   Spin down the cells, remove the supernatant, and resuspend in 1 mL of YPDA medium for a 40-minute to 2-hour recovery at 30°C (Optional for auxotrophic markers, but recommended for antibiotic selection) (Direct, High; PMID: 35781197, PMID: 26330622).

## Selection and Verification
*   **Plating:** Plate the cells on synthetic complete (SC) dropout agar media lacking the specific nutrient corresponding to the plasmid's selectable marker (e.g., SC-Ura, SC-Lys) (Direct, High; PMID: 26330622, PMID: 19074487).
*   **Incubation:** Incubate plates at 30°C for 2–4 days until colonies appear (Direct, High; PMID: 26330622, PMID: 19074487).
*   **Verification:**
    *   **Colony PCR:** Use verification primers that anneal across the junctions of the assembled fragments to confirm the correct structure (Direct, High; PMID: 19074487, PMID: 26330622).
    *   **Plasmid Recovery:** To confirm the sequence or perform complex verification, isolate yeast genomic DNA and transform it into *E. coli* to recover and amplify the circularized plasmid (Direct, High; PMID: 26330622, PMID: 19074487).
    *   **Restriction Analysis:** Perform restriction digestion on recovered plasmids to confirm the expected banding pattern (Direct, High; PMID: 19074487, PMID: 26330622).

[How do different yeast strains or mutant backgrounds like lig4Δ affect the efficiency and accuracy of in vivo gap repair?](#cta)

[What specific molar ratios of insert to vector are recommended for assembling plasmids with more than five DNA fragments?](#cta)

[How does the presence of repetitive sequences or high GC content in homology arms impact the fidelity of yeast gap repair cloning?](#cta)

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### Unverified Citations

The following sources failed to support their assigned claims after 3 verification rounds designed to ensure only high-confidence, relevant references are retained:

- **PMID:20300182** — *In budding yeast (*Saccharomyces cerevisiae*), in vivo gap repair—also known as gap-repair cloning (GRC) or DNA assembly...*  
  Failed: disease — The claim specifically asserts the efficiency and mechanism of GRC in budding yeast (Saccharomyces cerevisiae), but the cited paper focuses on the fission yeast Schizosaccharomyces pombe.
- **PMID:26330622** — *, 40–50 bp) are generally recommended to ensure high accuracy and colony yields*  
  Failed: conclusion — The claim recommends 40–50 bp for high accuracy, but the paper specifically describes and validates an efficient strategy using 30-bp homology regions.
- **PMID:26330622** — *Primers must be designed such that the 5' ends of the forward and reverse primers contain the 50 bp sequence homologous ...*  
  Failed: conclusion — The claim specifies designing primers with 50 bp of homology, but the paper's protocol is built around and validated using 30-bp homology regions.
- **PMID:26330622** — **   Add 5 µL of boiled, ice-chilled ssDNA (10 mg/mL carrier DNA) to the cell pellet*  
  Failed: conclusion — The paper specifies adding 2 µL of ssDNA, whereas the claim specifies 5 µL.
- **PMID:26330622** — **   Add 50–56 µL of DMSO (approximately 10% final concentration) and vortex briefly to resuspend the cells*  
  Failed: conclusion — The claim describes adding 50–56 µL of DMSO directly to cells, whereas this protocol incorporates 50 µL of DMSO into a 1050 µL master transformation buffer, resulting in a much lower final concentration (~5%) than the ~10% claimed.

I want to create plasmid through budding yeast in-vivo gap repair mechanism. My plan is to linearize my plasmid via PCR, and amplify my target gene with 50 bp homology to either end of the linearized

DNA Preparation and Design

High-Efficiency Transformation Protocol

Selection and Verification

Unverified Citations

DNA Design and Preparation

High-Efficiency Transformation Protocol

Selection and Verification

Unverified Citations