PROTOCOLS

In this page you will find the protocols that worked well for us.

Transformation Protocols

NEB transformation protocol

Materials

• DNA
• Competent cells
• SOC medium (SOB + Glucose) - room temperature
• Petri dish with antibiotic resistance - 37 ºC

Procedure

1. Thaw competent cells on ice.
2. Chill 5 ng of ligation mixture in a 1.5 ml microcentrifuge tube and then add 50 µL of competent cells. Do not vortex or mix.
3. Place the mixture on ice for 30 min (do not mix).
4. Heat-shock cells at 42 ºC for 30 s in a pre-heated water bath (works better than on a heating block) (do not mix).
5. Place on ice and add 950 µL of room temperature SOC medium to the tube (make sure that it is not contaminated).
6. Place tube at 37 ºC for 60 min. Shake or rotate (250 rpm). Warm selection plates to 37 ºC (not necessary, but increases efficiency).
7. Spread 50-100 µL of samples on plates (dependent on efficiency of the competent cells); incubate overnight at 37 ºC.

You can find the original NEB Transformation Protocol here.

iGEM Transformation Protocol

Materials

• DNA
• Competent cells
• SOC medium (SOB + Glucose)
• Petri dish with antibiotic resistance

Procedure

1. Thaw competent cells on ice.
2. Add 50 µL of thawed competent cells into a pre-chilled 2 mL tube.
3. Add 5 ng of resuspended DNA to the sample and pipet gently up and down a few times.
4. Keep the sample on ice for 30 min.
5. Heat-shock cells at 42 ºC for 60 s in a pre-heated water bath (works better than on a heating block).
6. Place samples on ice for 5 min (can be more).
7. Add 200 µL of SOC medium to the sample (make sure that it is not contaminated).
8. Incubate cells at 37 ºC for 2 hours with shaking or rotating. 2 hours recovery time after incubation helps in transformation efficiency.
9. Plate 20 µL and 200 µL of the sample on two plates and spread.
10. Incubate the plates at 37 ºC for 12-14 hours (agar side of the plate has to be up). Note: Incubating for too long will increase the occurrence of satellite colonies (especially if resistant to Ampicillin).

You can find the original iGEM Transformation Protocol here.

Materials

Procedure