E. coli Transformation

Long

1) Get 200ul aliquots of E. coli (DH5a for normal transformation or DE3 for expression) from -80C freezer and let thaw on ice.

2) Add DNA

For plasmid: 1ul DNA desired

For ligation: 10ul ligation reaction

-Also include a negative control with no DNA.

3) Incubate for 30min on ice.

4) Heat shock for 90sec at 42C.

5) Incubate for 1min on ice.

6) Add 1ml LB.

7) Agitate at 37C for 45min – 2hrs (1hr is good).

8) Spin down and pour off LB leaving ~100ul.

9) Resuspend cells in the ~100ul.

10) Plate the entire suspension on appropriate prewarmed plates.

11) Incubate at 37C overnight.

Short

Note: Use for amplifying AmpR plasmids only.

1) Get 200ul aliquots of E. coli (DH5a) from -80C freezer and let thaw on ice.

2) Aliquot 50ul of cells into tubes.

3) Add 1ul DNA desired.

4) Incubate on ice 5min-30min.

5) Plate on prewarmed LB-Amp plates. (Plate the whole thing--no spinning, no nothing).

6) Incubate at 37C overnight.

Blue/White Selection

-Used for plasmids like Bluescript.

-No insert=Blue. Insert=White.

-Do long transformation of ligations basically as described above.

-While cells are agitating,

A) Spread X-Gal and IPTG on plates.

X-Gal (50mg/ml): 20ul

IPTG (1M): 10ul

B) Incubate at 37C to dry (about 30min).