Materials and Reagents

1. 12 ml sterile plastic tubes
   
2. 15 ml sterile plastic tubes
   
3. Sterile plastic Petri dish plates (standard size; 100 x15 mm)
   
4. Sterile tips for micropippettes, 5-200 µl (for instance, Daslab, catalog number: 162001 )
   
5. Sterile tips for micropippettes, 100-1,000 µl (for instance, Daslab, catalog number: 162222 )
   
6. Sterile tips for micropippettes, 10 µl (for instance, Metler Toledo, catalog number: 17004280 )
   
7. Recipient Thermus strain, for instance, HB27 wild type (HB27/ATCC BAA-163 /DSM 7039)^{#
   #}Note: Other Thermus thermophilus strains such as NAR1 and HB8, have been defined as naturally competent, thus, harboring the competence proteins required for transformation (César et al., 2011). All Thermus spp. strains referred here are available at the DSMZ collection.
   
8. Kanamycin sulphate (Sigma-Aldrich, catalog number: K1377-5G )
   
9. Tryptone (Conda, catalog number: 1612 )
   
10. Yeast extract (Conda, catalog number: 1702 )
    
11. Sodium chloride (NaCl) (EMD Millipore, catalog number: 1.06404.1000 )
    
12. Agar (grade A) (Conda, catalog number: 1800 )
    
13. Thermus water (carbonated-rich mineral water)
    
14. DNA template: purified plasmids or genomic DNA** (harbouring the thermostable kat cassette)
    **Note: DNA quantity depends on the topology of the DNA to be transformed and the competence of the recipient strain.
    
15. TB liquid medium (see Recipes)
    
16. TB agar plates (see Recipes)
    

Equipment

1. 50 ml Erlenmeyer flask
   
2. Rotary shaker incubator reaching 70 °C (150 rpm) (Thermo Scientific, Thermo Scientific^TM, catalog number: SHKE 420HP )
   
3. Glass beads or a spreader
   
4. Autoclave device (CertoClav, model: EL 18L )
   
5. Spectrophotometer (Hitachi, model: U-2000 )
   
6. Pipette P2, 0.2-2 μl (Gilson, Pipetman classic^TM, catalog number: F144801 )
   
7. Pipette P20, 2-20 μl (Gilson, Pipetman classic^TM, catalog number: F123600 )
   
8. Pipette P100, 20-100 μl (Gilson, Pipetman classic^TM, catalog number: F123615 )
   
9. Pipette P200, 50-200 μl (Gilson, Pipetman classic^TM, catalog number: F123601 )
   
10. 70 °C heater (Gemini, model: Memmert UE400 )
    
11. Humid chambers
    Note: Tupperware devices are preferentially used. Humidified plastic bags are also valid. Addition of a paper towel, soaked on Thermus water and laid on the bottom of the chamber would enhance humidity maintenance. Ventilation should be avoided in order to conserve humidity. An example is provided in the photographs beneath (Figure 1).
    
    
    Figure 1. Humid chambers. Descriptive photographs of the humid chambers used to incubate Thermus plates. Place a piece of paper soaked with Thermus water and a bottle cap filled with the same water on the bottom of the chambers to maintain high humidity.

Procedure