Chapter_3___How_to_clone_a_gene_v2

Chapter_3___How_to_clone_a_gene_v2 - BasicMolecularBiology...

Info iconThis preview shows pages 1–7. Sign up to view the full content.

View Full Document Right Arrow Icon
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Basic Molecular Biology 3.1 What is cloning? ( key concept: propagation of a foreign gene in a vector) 3.2 Overview of the Procedures 3.3 Gene libraries ( key concept: each library insert is a unique clone ) 3.4 Hybridization ( key concept: probing for DNAs that will base pair with another DNA strand ) 3.5 Polymerase Chain Reaction (PCR) ( key concept: DNA synthesis between sets of hybridizing primers ) 3.6 Extraction and Purification of Nucleic Acids 3.6.1 Breaking up cells and tissues ( key concept: separation of cellular DNA, RNA, mRNA, protein, lipid ) Enzymes, Phenol-chloroform extraction, Ethanol precipitation, Gradient purification 3.6.2 Alkaline denaturation ( key concept: separation of bacterial plasmid DNA from bacterial chromosomal DNA ) 3.6.3 Column purification Size selection chromatography ( key concept: separation of DNA / RNA / protein based on size ) affinity chromatography ( key concept: cleaning DNA using DNA binding properties in salt or no salt ) 3.7 Detection and Quantitation of Nucleic Acids ( key concept: quantitation of DNA, RNA using spectroscopy) 3.8 Gel Electrophoresis Agarose and polyacrylamide gel electrophoresis ( key concept: separation of DNA based on size ) Electrophoretic mobility of nucleic acid form
Background image of page 2
3.1 What is Cloning? Cloning means using asexual reproduction to obtain genetically identical organisms Cloning extends to genes: introduction of a foreign gene into a bacterium that is replicated along with bacterium’s other DNA Cloned genes are identical in all replicating bacteria harboring the gene Figure 3.1 Comparison of bacterial cloning and gene cloning
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
3.2 Overview of the Procedures Bacteria take up DNA by transformation when subject to calcium and heat ( heat shock ) or electrical pulse ( electroporation ) DNA is only replicated by the host cell if it is inserted into the host chromosome or in vectors that are recognized by enzymes within the host cell as a substrate for replication Bacterial vectors are plasmids or bacteriophage Figure 3.1 Comparison of bacterial cloning and gene cloning
Background image of page 4
3.2 Overview of the Procedures A DNA insert cloned into a vector produce a recombinant DNA molecule that will replicated in all descendants of the initial transformant Bacteria divide rapidly, doubling every 20 minutes, producing 1 x 10 9 identical bacteria / ml in overnight culture Bacteria stop multiplying in a densely populated liquid culture because of limitations in nutrients including sufficient aeration with oxygen Exponential growth resumes after a small bacterial sample is added to fresh liquid media Figure 3.2 Basic outline of gene cloning
Background image of page 5

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
3.2 Overview of the Procedures Enzymes called restriction endonucleases break the DNA sugar- phosphate backbone at precise sites Cutting of both the inset and vector with the same restriction enzymes produces matching “ sticky ” (overlapping) or
Background image of page 6
Image of page 7
This is the end of the preview. Sign up to access the rest of the document.

Page1 / 25

Chapter_3___How_to_clone_a_gene_v2 - BasicMolecularBiology...

This preview shows document pages 1 - 7. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online