amplified Protocols

Category: PCR Protocols: AFLP PCR

Ulrich G. Mueller and L. LaReesa Wolfenbarger. Amplified fragment length polymorphisms (AFLPs) are polymerase chain reaction (PCR)-based markers for the rapid screening of genetic diversity. AFLP. TREE October 1999 - [Read AFLP genotyping and fingerprinting Review PDF]

Category: PCR Protocols: cDNA Synthesis Protocols

An oligodeoxynucleotide primer hybridized to mRNA is extended by an RNA-dependent DNA polymerase to create a cDNA copy that can be amplified by PCR.  Depending on the purpose of the experiment, the primer for first-strand cDNA synthesis can be specifically designed to hybridize to a particular target gene, or a general primer such as oligo(dT) can be used to prime cDNA synthesis from essentially all mammalian mRNAs - [Read Amplification of cDNA Generated by Reverse Transcription of mRNA Protocol]

Category: BACs Bacterial Artificial Chromosome Protocols

Protocol presents the amplification of insert end sequences from bacterial artificial chromosome clones using TAIL-PCR. The amplified products are suitable as probes for chromosome walking and genome mapping and as templates for direct sequencing. The protocol has been used in rice genome studies. - [Read Amplification of Insert End Sequences from BACs Clones by Thermal Asymmetric Interlaced PCR]

Category: PCR Protocols: AFLP PCR

Amplified Fragment Length Polymorphisms and Microsatellites: A phylogenetic perspective. Julian P. Robinson, Stephen A. Harris. What are AFLPs and how are they produced? How AFLPs have been used? Problems? Restriction Enzymes and Primers. AFLP Reproducib - [Read Amplified Fragment Length Polymorphisms and Microsatellites]

Category: PCR Protocols: AFLP PCR

An introduction to AFLP and fAFLP. Mark E. Berres, University of Wisconsin. Amplified fragment-length polymorphism (AFLP) or its fluorescent version (fAFLP) is a PCR-based fingerprinting technology. AFLP basically involves the restriction of genomic DNA - [Read An introduction to AFLP and fAFLP]

Category: PCR Protocols: PCR Cloning Protocols

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. - [Read Bidirectional Cloning of PCR Products Protocol]

Category: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

Protocol for cloning of amplified miRNA target sites into 3'-UTR of luciferase reporter vector. - [Read Cloning of Amplified miRNA Target Sites into 3'-UTR of Luciferase Reporter Vector Protocol]

Category: PCR Protocols: PCR Cloning Protocols

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. - [Read Cloning PCR Products by Addition of Restriction Sites to the Termini of Amplified DNA Protocol]

Category: PCR Protocols: PCR Cloning Protocols

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. - [Read Cloning PCR Products into T Vectors Protocol]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

The first step in competitive RT-PCR is the synthesis and purification of the synthetic competitor.  This is an RNA molecule designed to be reverse-transcribed and PCR-amplified with the same efficiency as the endogenous transcript of interest.  Once the competitor molecule has been prepared, as described in this protocol, competitive PCR can be carried out. - [Read Competitive RT-PCR: Preparation of Competitor RNA Protocol]

Category: DNA Protocols: DNA Sequencing Protocols

Direct sequencing from amplified bacterial and large insert cloned human or mouse genomic DNA via an improved MultiPlex PCR-based method. Includes: Preparing primers for MP-PCR; Amplification; PCR Product Clean-up; Sequencing. - [Read Direct Sequencing Using MultiPlex PCR-Based Method]

Category: PCR Protocols: PCR Cloning Protocols

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. - [Read Directional Cloning of PCR Products Protocol]

Category: DNA Microarray Protocols

This protocol describes the hybridization of a Cy labeled cDNA probe (mix of Cy3 and Cy5) onto coated slide spotted with PCR amplified cDNA. TIGR Microarray Protocols. - [Read Labeled Probe Hybridization Procedure]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

5 ml liquid lysates are prepared when a small amount of DNA from a large number of lambda clones is needed. The lysates can be made using 10- 20 ul of a stock lysate or a 100-fold amplified phage "macroplaque" as the inoculum. - [Read Liquid Phage Lysates Protocol]

Category: PCR Protocols: PCR Cloning Protocols

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. - [Read Molecular Cloning of PCR Products Protocol]

Category: DNA Protocols: DNA Sequencing Protocols: Big Dye Protocols

Protocol describes the purification, quantification, and subsequent sequencing of amplified DNA fragments using PCR. Excess nucleotides are removed from the initial PCR products using spun columns, and the products are quantified using fluorometry. - [Read Nonradioactive Cycle Sequencing of PCR-Amplified DNA Protocol]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

One step extraction for isolation of plant DNA. DNA suitable for amplification by PCR can be produced from leaf material smaller than 0.3 mm2 in less than 20 min & no tube changes. Method was tested on several plant species. Method was found to extract DNA that could be amplified without any further purification or treatment. The isolated DNA was amplified using a universal chloroplast primer set. The method was validated by comparing size of PCR products generated using standard DNA isolation. - [Read One-Step Isolation of Plant DNA Suitable for PCR Amplification]

Category: PCR Protocols: PCR Optimization

Procedure details the establishment of an amplification procedure for GC-rich sequences.  The DNA fragments of interest are amplified in the presence of either 5% DMSO, 1 M betaine, 2 M betaine, 1 M betaine, and 5% DMSO; 2 M betaine and 5% DMSO; 0.4 M tetramethylene sulfone; or without any of the enhancers. - [Read PCR Amplification of Highly GC-Rich Regions Protocol]

Category: Genetics and Genomics: Genotyping Protocols: PCR Genotyping

Using molecular marker technology in studies on plant genetic diversity. DNA-based technologies: PCR-based technologies Amplified fragment length polymorphisms (AFLPs. Includes: AFLP technology, step by step; DNA digestion and ligation; PCRs and detection; Summarising the technology. - [Read PCR-Based Technologies Amplified Fragment Length Polymorphisms (AFLPs)]

Category: Cloning Protocols

Protocol describes how to use proteinase K to destroy thermostable enzymes and to purify amplified DNA in preparation for cloning. - [Read Purification of PCR Products in Preparation for Cloning Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols

This protocol describes how to use proteinase K to destroy thermostable enzymes and to purify amplified DNA in preparation for cloning. - [Read Purification of PCR Products in Preparation for Cloning Protocol]

Category: PCR Protocols: PCR Cloning Protocols

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. - [Read Rapid Characterization of DNAs Cloned in Prokaryotic Vectors Protocol]

Category: Nucleic Acid Purification Protocols

In this protocol, ultrafiltration through Centricon or Microcon concentrators is used to remove unused primers, primer-dimers, and NTPs from preparations of amplified DNA. - [Read Removal of Oligonucleotides and Excess dNTPs from Amplified DNA by Ultrafiltration Protocol]

Category: DNA Protocols: Genomic DNA Library Protocols

Amplified cDNAs containing functional 3' and 5' splice sites are selected by digestion with BstXI and cloned into a Bluescript vector. - [Read Reverse Transcriptase-PCR for Exon Trapping and Amplification]

Category: PCR Protocols: PCR Cloning Protocols

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. - [Read Ribocloning: DNA Cloning and Gene Construction Using PCR Primers Terminated with a Ribonucleotide]