Materials and Reagents

1. Recipient strain [ATCC2001, HTL or clinical isolates (Schwarzmuller et al., 2014)]
   
2. DNA deletion construct/transforming DNA
   
3. Sterile water (double distilled)
   
4. Bacto™ peptone (BD Biosciences, catalog number: 211820 )
   
5. Bacto™ yeast extract (BD Biosciences, catalog number: 212720 )
   
6. Bacto™ agar (BD Biosciences, catalog number: 214030 )
   
7. Glucose (Merck KGaA, catalog number: 108337 )
   
8. Lithium acetate dehydrate (LiAc) (Sigma-Aldrich, catalog number: L6883 )
   
9. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 472301 )
   
10. Polyethylene glycol (PEG 3350) (Sigma-Aldrich, catalog number: P4338 )
    
11. Nourseothricin-dihydrogen sulfate (Werner BioAgents, catalog number: 5.0 )
    
12. ssDNA (Sigma-Aldrich, catalog number: D1626 )
    
13. YPD media (see Recipes)
    
14. Solid selective media (see Recipes)
    

Equipment

1. Deep well plate (96-well) (Nunc^®, catalog number: 732-2662 )
   
2. Reservoir (autoclavable) (VWR International, catalog number: 6130466 )
   
3. Multichannel pipette (200 µl) (Brandt Transferpette, WU2160016)
   
4. Culture flasks with baffles
   
5. Centrifuge (50 ml tubes) (Eppendorf, catalog number: 5702R )
   
6. Centrifuge (1.5 ml tubes) (Eppendorf, catalog number: 5417R )
   
7. Rotary shaker for culture flasks (New Brunswick Scientific, catalog number: innova44 )
   

Procedure