Lab Techniques. James Chappell & Cheuk Ka Tong. Contents Page. Restriction Enzymes Gel Electrophoresis Blotting techniques-Southern, Northern and Western DNA sequencing Polymerase Chain Reaction (PCR) Recombinant DNA Gene Cloning References/ Recommended Reading. Restriction Enzymes.
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James Chappell & Cheuk Ka Tong
Two key terms
"The work on restriction nucleases not only permits us easily to construct recombinant DNA Molecules and to analyze individual genes but also has led us into the new era of synthetic biology where not only existing genes are described and analyzed but also new gene arrangements can be constructed and evaluated"
‘Nobel prizes and restriction enzymes’ in GENE (1971)
Mobility = Charge + Molecular Dimensions
- identification of various parts in natural organisms,
This is essentially the monomer of DNA. Polymerization of nucleotides occurs by condensation reaction of a 5’ phosphate to a 3’ hydroxyl group
There is no 3’hydroxyl group to allow polymerization.
- Target sequence
- A pair of primers
- dNTPs (ATGC)
- DNA polymerase
- Strand separation (95ºC)
- Hybridization of primers (54ºC)
- DNA synthesis (72ºC)
- A vector can replicate autonomously in an appropriate host.
- Plasmids and phage λ are common vectors for cloning in E.coli.
Basis for selection of cells containing recombinant DNA.
- Molecular Biology of the Cell (4th Edition), Alberts, Johnson, Lewis, Raff, Roberts, Walter, Garland Science
2. Chapter 6- Exploring Genes
-Biochemistry (5th Edition), Berg, Tymoczko, Stryer, Freeman