Basic Tools: Recombinant DNA Techniques Cut Purified DNA with Restriction Enzymes Transform E. coli Purified plasmid DNA Various restriction enzymes T4 DNA ligase E. coli bacteria

DNA Oligonucleotide Chemical Synthesis Synthesis of short (1 to 100 bp) single-stranded DNA molecules of defined sequence using solid phase chemistry on a DNA synthesizer Used for sequencing, PCR, custom gene synthesis

The Polymerase Chain Reaction (PCR)‏ Allows amplification of a target sequence from biological sources Oligonucleotides do not have to match the template exactly Used for cloning and site-directed mutagenesis Amplification of DNA with a thermostable DNA polymerase, the monomeric precursors of DNA (dNTPs), and two short (20 bp) single-stranded oligonucleotides using an applied temperature program

Cloning Vectors Pre-PCR ApproachModern Single-Gene Cloning Vectors Proc Natl Acad Sci U S A Dec;82(23):7870-3