cloning with plasmids
DESCRIPTION
Cloning with Plasmids. 1973 Genetic Engineering Invented . MARKER. Cloning: Digestion. TARGET GENE. SOURCE DNA. MARKER. Cloning: Digested Fragments. TARGET GENE. MARKER. Cloning: Ligation. DNA LIGASE. TARGET GENE. MARKER. Cloning: Recombinant Plasmid. TARGET GENE. - PowerPoint PPT PresentationTRANSCRIPT
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