Cloning with Plasmids

1973 Genetic Engineering Invented

Cloning: Digestion

TARGET GENE

SOURCE DNA

MAR

KER

MAR

KER

Cloning: Digested Fragments

TARGET GENE

MAR

KER

Cloning: Ligation

TARGET GENE

DNA LIGASE

MAR

KER

Cloning: Recombinant Plasmid

TARGET GENE

Cloning: Transformation

E. coli

Cloning: Competence

E. coli

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+ Ca2+

Ca2+

Ca2+

Ca2+ Ca2+Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+ Ca2+Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+ Ca2+

Cloning: Preparing Competent Cells

• inoculate culture with a 1/100 dilution of an overnight• grow to an OD600 of 0.2• transfer to 50 ml Falcon tubes, chill on ice 10'.• pellet in Beckman kneewell centrifuge, 3K, 10'.• resuspend in 12.5 ml 100mM MgCl2 (This is best done by initially

resuspending the cells in 1 ml, using a P1000 and then adding an additional 11.5 ml.)

• pellet• resuspend in 25ml 100mM CaCl2 (1ml then 24).• incubate on ice for 20 minutes• pellet• resuspend in 0.5 ml 85mM CaCl2 and 15% glycerol• snap freeze in liquid nitrogen, keep @ -70°C.

Cloning: Competence

E. coli

Cloning: Transformation

E. coli

Cloning: Transformation

E. coli

Cloning: TransformationAfter ligations have gone for one hour, you will do the following in order to transform the competent cells:

1) Thaw competent cells and quickly add 150 µl of these cells to each ligation tube. Mix gently. Leave on ice for 20 minutes.

2) Heat shock cells by placing tubes in a 42°C water bath for 90 seconds.

3) Add 0.8 ml L broth and incubate at 37°C with gentle shaking for 1 hour.

Cloning: Plating

plasmid plasmidligase

plasmidkan gene

ligase

amp

kan

Today’s Activities

• Set up ligation • Set up digestions to complete map and for

blot (use 2x volume for blots)• Transform ligations into competent cells• Plate transformations onto amp and kan

plates