3 June 2009

Aim
 * Transformation

Materials:

•TE Buffer

•Plasmids from Kit Plates 1 & 2

•Competent E.coli

•NZY Medium

•Ampicllin, Kanamycin, Tetracyclin

•Agar Plates

•Eppendorf (labelled)

Methods：


 * Transformation Protocol


 * 1) Hydrate plasmids by 10µL TE buffer.


 * 1) Add 5µL plasmid solution into labelled eppendorf, together with 15µL TE buffer. (Total Vol = 20µL)


 * 1) Take 5µL from the 4-fold dilution in step 2, add into the competent E.coli.


 * 1) Incubate on ice for 30 min. (minimum) [Actual incubation time in lab: >1 hr]


 * 1) Incubate at 42°C for 45 sec.


 * 1) Incubate on ice for 2 min. (minimum)


 * 1) Add 300µL of NZY medium.


 * 1) Incubate with shaking at 37°C for 1 hr.[Actual incubation time in lab: 40 min]


 * 1) Spin the incubated E.coli suspension for 3min at maximum speed.
 * 2) Discard about half of the liquid, and then re-suspend the E.coli solution.
 * 3) Spread the suspension onto selective agar plates.
 * 4) Incubate at 37°C overnight.


 * Making Agar Plates


 * 1) Defreeze agar in microwave oven, 1 min interval.


 * 1) Check and shake until completely melting.


 * 1) Add 100µL antibiotic into 50ml agar when it cools down to 50°C.


 * 1) Pour the plates.