Team:UCL London/NZY+ Broth (per Liter)

NZY + Broth

(per litre)

10 g of NZ amine (casein hydrolysate)

5 g of yeast extract

5 g of NaCl

Add deionized H2O to a final volume of 1 liter. Adjust to pH 7.5 using NaOH.

Autoclave.

Add the following filer-sterilized supplements prior to use:

12.5 ml of 1 M MgCl2 12.5 ml of 1 M MgSO4 20 ml of 20% (w/v) glucose (or 10 ml of 2 M glucose) Transformation:

Materials:

Plasmids DNA

Competent E.coli

NZY Medium

LB buffer

Ampicllin, Kanamycin, Tetracycline

Agar Plates

Eppendorf

Method

1. Punch the appropriate well with a clean tip.

2. Soak the spots in 10µL of TE that has been warmed to 50ºC for 20 minutes. At this time take the competent cells from the -80º freezer and start defrosting on ice.

3. Add 4µL of DNA in TE to 100µL of competent cells.

4. sit on ice for 30 minutes

5. Heat shock at 42ºC for 60 sec in water bath.

6. Recover on ice for 5 min.

7. Add 300µL of NZYX medium.

8. Incubate with shaking at 37°C for 2hrs. (1hr)

9. Spin the incubated E.coli suspension for 3min at maximum speed. (Optional)

10. Discard about half of the liquid, and then re-suspend the E.coli solution. (Optional)

11. Spread the suspension onto selective agar plates.

12. Incubate at 37°C overnight.

13. Pick a colony into 2ml selective LB (Add 100µL antibiotic to 50mL LB medium). Incubate 37°C in shaker overnight.