PCR Cloning Protocols

Bidirectional Cloning of PCR Products Protocol Protocol is for bidirectional, blunt-end cloning of DNA fragments.  The target DNA is PCR amplified and 3'-extensions are polished with Pfu DNA polymerase.  The amplicon is ligated to a blunt-ended plasmid DNA, and the products of the ligation reaction are used to transform competent Escherichia coli.  A restriction enzyme is added to the ligation reaction to relinearize any self-religating vector DNA.

Blunt-end Cloning of PCR Products Protocol Protocol for blunt-end cloning of PCR products. Incubation of a blunt-end ligation reaction in the presence of an excess amount of an appropriate restriction enzyme can dramatically increase the yield of recombinant plasmids.  The role of the restriction enzyme is to cleave circular and linear concatemers at restriction sites that are re-formed when linear, blunt-ended plasmid molecules ligate to themselves.  I

Cloning PCR Products by Addition of Restriction Sites to the Termini of Amplified DNA Protocol Pairs of oligonucleotide primers used in PCR are often designed with restriction sites in their 5' regions.  In many cases, the sites are different in the two primers.  In this case, amplification generates a target fragment whose termini now carry new restriction sites that can be used for directional cloning into plasmid vectors.  The purified fragment and the vector are digested with the appropriate restriction enzymes, ligated together, and transformed into E. coli.

Cloning PCR Products into T Vectors Protocol This method of direct cloning takes advantage of the unpaired adenosyl residue added to the 3' terminus of amplified DNAs by Taq and other thermostable polymerases.

Directional Cloning of PCR Products Protocol Protocol is for directional blunt-end cloning of DNA fragments.  The target DNA is PCR-amplified, 3'-extensions are polished with Pfu DNA polymerase, and the amplicon is ligated to a blunt-ended plasmid DNA.  The products of the ligation reaction are used to transform competent Escherichia coli.  A restriction enzyme is added to the ligation reaction to relinearize any self-religating vector DNA.

Isolating the Ends of Genomic DNA Fragments Cloned in High-capacity Vectors Protocol describes the use of vectorette PCR and single-site PCR to amplify the terminal sequences of genomic sequences cloned in high-capacity vectors such as PACs and YACs.

Molecular Cloning of PCR Products Protocol The efficiency of direct cloning of PCR products can be improved by generating suitable ends on the amplified fragments.  This protocol describes the strategies for generating and manipulating suitable ends on the PCR fragments.

Rapid Characterization of DNAs Cloned in Prokaryotic Vectors Protocol In this protocol sequences cloned in standard bacteriophage or plasmid vectors are amplified in PCRs containing primers targeted to flanking vector sequences.  The amplified fragments can be analyzed by gel electrophoresis, DNA sequencing, and/or restriction mapping.  Many colonies or plaques can be assayed simultaneously.

Ribocloning: DNA Cloning and Gene Construction Using PCR Primers Terminated with a Ribonucleotide Protocol exploits the discovery that Rnase A can efficiently cleave at single rC or rU bases embedded in double-stranded DNA.  Entire plasmid vectors are amplified using long, high-fidelity PCR with riboprimers, which carry a single rC residue at their 3' end.  Target DNA is amplified using similar primers, which also end in a rC residue.