DH5α competent cells preparation

1. The day before the preparation, make overnight starters with 100 µL of bacterial cells diluted in 5 mL of LB in a 50 mL Erlenmeyer.
   
2. Prepare buffers 1 and 2 and put them on ice. Put Eppendorf tubes and 50 mL Falcon tubes, and p1000 tips at -80°C for future use.
   
3. The next day, recover your starters and seed 1/60 bacterial cells in 200 mL of LB, then incubate at 37°C and 180 rpm.
   
4. Keep measuring the OD600. When it's between 0,4 and 0,6, recover your cells an pool them in cold 50 mL Falcon tubes.
   
5. Centrifuge tubes at 4000 rpm and 4°C for 10 minutes.
   
6. Discard supernatant and resuspend each Falcon tube in 20 mL of buffer 1. Afterward, centrifuge at 3500 rpm and 4°C for 5 minutes.
   
7. Discard supernatant and resuspend each Falcon tube in 8 mL of buffer 2.
   
8. Take 100 µL aliquots in Eppendorf tubes and stock them at -80°C.
   

Transformation

1. Recover the needed number of competent cells tubes, adding an additional negative control tube.
2. Add DNA (1µL) into the competent cells tubes.
3. Incubate tubes on ice for an hour.
4. Incubate tubes at 42°C for 2 minutes for thermal choc.
5. Leave tubes on ice for 5 minutes.
6. Leave tubes at room temperature for 5 minutes.
7. Add 1 mL of LB per tube, then incubate for 1 hour at 37°C and 180 rpm.
8. Spread cells on Petri dishes and incubate overnight at 37°C.