Molecular Biology

Chapter 4/5 Outline

Restriction enzymes:

∑ Cutting characteristics and sequences (T4.1); restriction-modification system (F4.1); Cohen and Boyer experiment (F4.2).

Amplifying DNA and RNA:

∑ Plasmid vectors - pBR322 derivatives (F4.3, F4.6; F4.11); ligation (F4.7); cloning sites; insert selection.

∑ Phage vectors - lambda phage (F4.8); cosmids; genomic libraries; infection; plaques.

∑ PCR (F4.12).

∑ Generating cDNA(F4.13).

∑ RT-PCR (F4.15); Rapid Amplification of cDNA Ends (RACE)(F4.16).

Expressing proteins from cloned DNA:

∑ Expressing proteins from clones (F4.18); vectors for E. coli; purifying fusion proteins (F4.19).

∑ Eukaryotic expression vectors; baculovirus (F4.22).

Separating DNA fragments:

∑ Gel electrophoresis(F5.1;F5.2); agarose and acrylamide (F5.4).

Identifying specific DNA fragments and RNAs:

∑ Transferring DNA and RNA to membranes (F5.12); Southern hybridization; DNA probes (random oligo labeling).

∑ DNA sequencing (F5.18).

Mapping/quantifying transcripts:

∑ Primer extension (F5.30).

∑ Northern blots (F5.16).

Determining transcription rates:

∑ Run-on transcription (F5.33).

Detecting DNA-protein interactions:

∑ Mobility shift assays (F5.36).

∑ DNase footprinting (F5.37).