cDNA Library Construction Protocols

Clone Genes From a Phage Library Protocol Protocol for cloning genes from a phage library. Includes: Titer and plate out phage; Lift plaques onto filters and prepare them for screening; Make a probe; Hybridize the probe to the filters; Wash the filters and expose to film; Purify putative plaques; Excise plasmid from the desired phage.

Generation of a Library of Randomly Overlapping DNA Inserts Protocol Shotgun sequencing of a large segment of DNA involves random fragmentation of the target region into smaller segments that are subsequently cloned into a bacteriophage M13 vector. The goal is to create a library of overlapping clones that provide at least fivefold coverage over the entire length of the target fragment.

Lambda Methods Media and Solutions Information on lambda methods media and solutions.

PCR-Based Screening of DNA Libraries Protocol PCR can be used to identify rare DNA sequences in DNA libraries by increasing the abundance of a particular sequence. This is accomplished by subdividing the original library into pools of decreased complexity and screening each pool or group of pools for a given DNA sequence.

Screening cDNA Libraries Constructed in Eukaryotic Expression Vectors Protocol Two methods of screening pools of cloned cDNAs are discussed: transfection into mammalian cells and injection into Xenopus oocytes.

Screening Expression Libraries Constructed in Bacteriophage Lambda Vectors Protocol An expression library constructed in a bacteriophage {lambda} vector is plated on an appropriate E. coli strain in the absence of isopropylthio-ß-D-galactoside (IPTG). After 2-4 hours, the plates are moved to 37°C (to stabilize any fusion proteins that are temperature sensitive), and filters impregnated with IPTG are laid on top of the developing plaques.

Shotgun Library Construction Protocol Protocol for shotgun library construction.

Synthesis of DNA Libraries: Use of PCR to Analyze Ligation Substrates and Products A vector that lacks inserts can significantly contaminate a DNA library. This contamination can occur either from self-ligation of single-cut vector or from uncut circular starting plasmid.

Use of PCR for Quality Control of a Peptide DNA Library Protocol Protocol describes the use of PCR to characterize a peptide library encoded in a plasmid vector. In this example, the library was obtained by transforming bacteria with the ligation reaction at the end of Use of PCR to Prepare a Double-Stranded DNA Library Encoding Random Peptides.

Use of PCR to Prepare a Double-Stranded DNA Library Encoding Random Peptides Protocol PCR is used as a preparative tool for the synthesis of a high-complexity double-stranded DNA library. In the example presented here, a mixture of synthetic oligonucleotides is used to synthesize a random peptide NNK library, where K is either T or G. The exclusion of A and C nucleotides at the third position decreases the occurrence of stop codons but still allows codons for all 20 amino acids.