Transformation of plasmid DNA to competent E. Coli cells

1. Thaw competent cells on ice. 20–200µL per tube
2. Add max. 1-3µL of plasmid
3. Mix very gently!
4. Incubate the tubes on ice for 30 min
5. Heat shock the cells for 45 sec to 2 min at 42°C
6. Place the tubes immediately on ice for at least 2 min
7. Add 800µL of SOC medium to each tube
8. Incubate for 1 hour at 37°C and shake vigorously
9. Spin down briefly and remove most supernatants
10. Resuspend cell pellet with the rest SOC medium in the tube by pipetting
11. Plate out the suspension on a LB agar plate containing the appropriate antibiotic. Incubate the plates overnight at 37°C

BACKBONE

Vector backbone pFLAG-CMV

Backbone manufacturer  Sigma

Backbone size w/o insert (bp)4646

Vector type  Mammalian Expression

GROWTH IN BACTERIA

Bacterial Resistance(s)  Ampicillin

Growth Temperature  37°C

Growth Strain(s)  DH5alpha

Copy number  High Copy

GENE/INSERT

Gene/Insert name  bJunVN173

Alt name  c-Jun(257-334)-Venus(1-172)

Species  R. norvegicus (rat); Aequorea Victoria

Insert Size (bp)  774

Mutation  c-Jun(257-338)-Venus(1-172)

Tag / Fusion Protein  FLAG (N terminal on backbone)

CLONING INFORMATION

Cloning methodRestriction Enzyme

5′ cloning siteEcoRI (not destroyed)

3′ cloning siteBamHI (not destroyed)