Figure 1. Flowchart of our method

Materials and Reagents

1. Plasmids/nucleic acids
   1. Plasmid pSU32 (http://www.iac.saga-u.ac.jp/lifescience/su32/index.htm) (Figure 2)
      
      
      Figure 2. Plasmid pSU32
      
      
   2. E. coli plasmid vector containing the Amp^r gene (bla gene) (e.g., pUC, pBluescript, and the other many types of commonly-used plasmid vectors)
      
   3. DNA fragment to be cloned
      
   4. PCR primers (the pSU30-14 and pSU30-23 pair for the preparation of the conversion cassette SU32, and primer pair for the preparation of the DNA fragment to be cloned)

      the primer pair for the preparation of the conversion cassette SU32
      pSU30-14	5'-CAGGTGGCACTTTTCGGGGAAATGTG-3'
      pSU30-23	5'-ATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA
      	AGTTTTAAATCAATCTAAAGTATATATGAGTAAACT-3'
      Example: Primer pair for the preparation of the DNA fragment to be cloned (in this case, the GFPuv gene was cloned into pUC19 plasmid. The crossover regions (the sequences to be joined) are underlined).
      pUCGFPF	5'-GAGCGGATAACAATTTCACACAGGAAACAGCTATGGCTAGCA
      	AAGGAGAAGAACT-3'
      pUCGFPR	5'-TTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTTATTTG
      	TAGAGCTCATCCA-3'

      
      
2. Kits
   5. (Optional) PCR Purification kit
      In our paper (Goto and Nagano, 2013), we used the MonoFas DNA Purification Kit I (GL Sciences, Tokyo, Japan). However, this kit currently does not work well in our laboratory. Instead, we are now using the NucleoSpin Gel and PCR Clean-up kit (MACHEREY-NAGEL, Düren, Germany).
      
   6. Plasmid DNA purification kit for E. coli
      We used a Mini-M Plasmid DNA Extraction System (Viogene, Taipei, Taiwan) (Goto and Nagano, 2013).
      
   7. Suspension buffer (in the case of Mini-M Kit, we use 200 μl of MX1 buffer)
      
      
3. Enzymes
   8. Proofreading DNA polymerase such as Prime STAR GXL DNA Polymerase (TAKARA BIO, Ohtsu, Japan)
      
   9. Restriction enzymes (New England Biolabs)
      
      
4. Cells
   10. Yeast cells (commonly used strains containing the genotype trp1, such as YPH499)
       
   11. Electro-competent E. coli cells
       We used the E. coli HST08 Premium Electro-Cells (TAKARA BIO, Ohtsu, Japan) (Goto and Nagano, 2013). However, these cells currently do not work well in our laboratory. Instead, we are now making electrocompetent cells ourselves according to Molecular Cloning (4^th edition, pages 177-182).
       
       
5. Plates
   12. LB agar containing 100 μg/ml ampicillin (Molecular Cloning, 4^th edition, page 1100)
       
   13. Synthetic complete plates lacking tryptophan (The Gietz Lab, University of Manitoba, http://home.cc.umanitoba.ca/~gietz/ or Gietz and Woods, 2002)
       
   14. Ampicillin (sodium salt)
       
       
6. Others
   15. Plating beads
       
   16. Acid-washed glass beads (350-500 μm)
       We are making acid-washed glass beads ourselves. However, Acid-washed glass beads (425–600 μm) can be obtained from SIGAMA-ALDRICH (catalog number: G8772).
       
   17. Agarose gel (Molecular Cloning, 4^th edition, pages 94-98)
       
   18. Materials and Reagents required for yeast transformation (The Gietz Lab, University of Manitoba, http://home.cc.umanitoba.ca/~gietz/ or Gietz and Woods, 2002)
       

Equipment

1. Thermal Cycler
   
2. Agorose gel electrophoresis device
   
3. Centrifuge
   
4. Optional: Picofuge
   
5. Incubators (30 °C and 37 °C)
   
6. Vortex machine
   
7. Optional: FastPrep FP100A (MP Biomedical)
   
8. Heat block (70 °C)
   
9. Electroporation device and cuvette
   
10. Polyacrylamide gel electrophoresis devices
    
11. Glass beads (350-500 μm)
    

Procedure