ABSTRACT
Blunt-Ended cDNAs ..................................................................................228 12.2.2.7 In Vitro Packaging of Ligated DNA .......................................................... 231
12.2.3 Titering and Ampli cation of the Packaged Phage .................................................. 232 12.2.4 Large-Scale Ligation and In Vitro Packaging .......................................................... 232 12.2.5 Immunoscreening of a Lambda Expression cDNA Library .....................................234 12.2.6 Troubleshooting Guide for Immunoscreening.......................................................... 237 12.2.7 Screening a cDNA Library Using Labeled DNA Probes ......................................... 237
12.2.7.1 Screening of a cDNA Library by a 32P-Labeled Probe .............................. 237 12.2.7.2 Screening of a cDNA Library Using a Nonradioactive Probe ................... 237
12.2.8 Reagents Needed ...................................................................................................... 237 12.3 Construction of a cDNA Library by Subtractive Hybridization Techniques........................ 241
12.3.1 Synthesis of the First-Strand cDNA .........................................................................242 12.3.2 Removal of the mRNA Template .............................................................................242 12.3.3 Hybridization of cDNA to mRNA ............................................................................242 12.3.4 Separation of cDNA/mRNA Hybrids from Single-Stranded cDNA
by Hydroxyapatite Chromatography ......................................................................... 243 12.3.5 Preparation of a cDNA Library from the Subtracted First-Strand cDNA ................ 243 12.3.6 Reagents Needed ......................................................................................................245
12.4 Construction of Fractional cDNA Libraries Using Xenopus Oocytes as an Expression System .......................................................................................................245 12.4.1 Reagents Needed ......................................................................................................250
12.5 cDNA Cloning and Analysis by the Polymerase Chain Reaction ........................................ 251 12.5.1 Selection of Oligonucleotides ................................................................................... 251 12.5.2 General Ampli cation of Double-Stranded DNA by PCR ...................................... 251 12.5.3 cDNA Cloning by RT-PCR .......................................................................................254 12.5.4 Analysis of Gene Expression by Semiquantitative PCR ..........................................256 12.5.5 Reagents Needed ...................................................................................................... 259
References ......................................................................................................................................260
