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Three important features1. Cloning site2. Ori-an origin of replication3. A selectable marker (ampr)
pGEM-3Z
Cloning foreign DNA into a plasmid vector
Alkaline phosphatase-removes 5’ phosphate (P) groups of DNA molecules; BAP is more stable but less active than CIP
T4 DNA ligase –joins 5’ phosphate (P) groups of DNA molecules to 3’ hydroxyl (OH) groups of DNA
Some antibiotics commonly used as selective agents
Antibiotic Description
Ampicillin (Amp) Inhibits bacterial cell wall synthesis; inactivated by -lactamase, which cleaves the -lactam ring of amp
Hygromycin B (HygB)
Kanamycin (Kan) Binds to 30S ribosomal subunit and inhibits protein synthesis; inactivated by a phosphotransferase
Neomycin (Neo) Binds to 30S ribosomal subunit and inhibits protein synthesis; inactivated by a phosphotransferase
Streptomycin (Str)
Tetracycline (Tet) Binds to 30S ribosomal subunit and inhibits protein synthesis; tetr gene encodes a protein which prevents transport of tet into the cell
Genomic library
construction
Screening a genomic library using DNA hybridization to a
(radio-)labeled DNA probe
Note: a cDNA is commonly (radio-)labeled
and used as a DNA probe to screen a genomic library
Production of a (radio-)labeled DNA probe by the random primer method [uses the Klenow fragment of DNA polymerase]
5’
5’5’
3’
3’3’
The first step in making a cDNA library: Purification of
polyadenylated mRNA using oligo(dT)-
cellulose
Note: selection of the proper source (organ, tissue) of the RNA is
critical here!
Complementary DNA or cDNA cloning:cDNA library construction
Note: ds cDNAs are typically placed in a cloning
vector such as bacteriophage lambda ()
or a plasmid
There are several possible ways to screen a cDNA library
• Using a DNA probe with a homologous sequence (e.g., a homologous cDNA or gene clone from a related species)
• Using an oligonucleotide probe based on a known amino acid sequence (requires purification of the protein and some peptide sequencing)
• Using an antibody against the protein of interest (note: this requires use of an expression vector)
• Plus/minus or differential screening (the least specific way)
Screening a cDNA library using DNA
hybridization to a (radio-)labeled
DNA probe
Screening a cDNA library with a labeled oligonucleotide probe based on a known peptide sequence
Using polynucleotide kinase and-32P-labeled ATP to radiolabel oligonucleotide probes
Immunological screening of an expression cDNA library with a primary antibody and labeled secondary antibody;
note the label is often an enzyme label like alkaline
phosphatase or horseradish peroxidase, but it can also
be 125I
Note: see also MCB Chapter 9 for a related animation