cDNA Library Protocols

This stage achieves four goals: polishing the ends of double-stranded DNA, ligation of synthetic linkers or adaptors, digestion of the attached linkers to create cohesive termini, and preparing the cDNA for cloning. - [Read Attachment of Linkers or Adaptors for Construction of cDNA Libraries]

Protocol for construction of cDNA library, stage 5: Fractionation of cDNA by Gel Filtration through Sepharose CL-4B. - [Read Construction of cDNA Libraries Stage 5: Fractionation of cDNA by Gel Filtration through Sepharose]

Here, the DNA-RNA hybrids synthesized in Stage 1 are converted into full-length double-stranded cDNAs. The primers for synthesis of second-strand cDNA are created by RNase H, which introduces nicks into the RNA moiety of the cDNA-mRNA hybrids. E. coli DNA polymerase I extends the newly created 3'-hydroxyl termini, using the first-strand cDNA as a template. - [Read Construction of cDNA Libraries Protocol]

The goal of this stage is to introduce methyl groups that will modify and protect naturally occurring EcoRI sites in the double-stranded cDNA. - [Read Construction of cDNA Libraries Protocol.]

The final stage of cDNA library construction involves optimizing ligation of the size-fractionated cDNA to bacteriophage {lambda} arms, followed by packaging and plating of the recombinant bacteriophages. - [Read Ligation of cDNA to Bacteriophage lambda Arms for the Construction of cDNA Libraries]

Protocol for RNA isolation for cDNA library construction. - [Read RNA Isolation for cDNA Library Construction Protocol]

A cDNA library constructed in a plasmid expression vector of the pUC, pUR, or pEX series is plated on agar medium and then replicated onto filters, which are transferred to plates containing IPTG. After 2-4 hours of induction, the colonies are lysed with chloroform and then screened with appropriate antibodies. - [Read Screening Expression Libraries Constructed in Plasmid Vectors Protocol]

Protocol describes the generation of cDNA probes from poly(A)+ mRNA using random oligonucleotide primers. Probes of this type are used for differential screening of cDNA libraries. - [Read Synthesis of cDNA Probes from mRNA Using Random Oligonucleotide Primers Protocol]

Protocol for the construction of construction of cDNA libraries by synthesis of first-strand cDNA catalyzed by reverse transcriptase. - [Read Synthesis of First-strand cDNA Catalyzed by Reverse Transcriptase for Construction of cDNA Libraries]