fragment Protocols

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

Protocol describes large fragment DNA replication by Adenovirus proteins using TP-DNA as template. - [Read Adenovirus DNA Replication Assay Protocol]

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: 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

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: Cloning Protocols: Mutagenesis Protocols

This approach can be used to delete a fragment of any lenth or to introduce point mutations into a DNA seqence. - [Read Creating a Deletion by PCR Splicing Protocol]

Category: Plant Biology Protocols: Plant Genetics Protocols

Virus-induced gene silencing (VIGS) uses a virus to deliver a sequence from a gene of interest into a host plant. The virus carrying the fragment of the gene of interest must be capable of replication if dsRNA is to be produced. One or two leaves are inoculated with Agrobacterium strains carrying the VIGS vector possessing the gene fragment. The virus then replicates and spreads throughout the plant, mediating silencing. - [Read Delivery of dsRNA into Plants by VIGS Methodology]

Category: DNA Protocols: DNA Sequencing Protocols: Dideoxy Mediated Sequencing Protocols

Protocol for dideoxy-mediated sequencing reactions using the Klenow fragment of E. coli DNA polymerase I and single-stranded DNA templates. - [Read Dideoxy-mediated Sequencing Reactions Using the Klenow Fragment of E. coli DNA Polymerase]

Category: E Coli Protocols: E coli Nucleic Acid Protocols

Protocol for dideoxy-mediated sequencing reactions using the Klenow Fragment of E. coli DNA polymerase I and single-stranded DNA templates. - [Read Dideoxy-mediated Sequencing Reactions Using the Klenow Fragment of E. coli DNA Polymerase I]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

Protocol for direct retrieval of DNA fragments from pulsed-field gels. A gel slice containing a fragment of DNA resolved by pulsed-field gel electrophoresis is treated with agarase.  The released DNA can be used as a substrate for ligation or restriction without further purification. - [Read Direct Retrieval of DNA Fragments from Pulsed-field Gels Protocol]

Category: Cloning Protocols: Vector Protocols

Directional cloning requires that the plasmid vector be cleaved with two restriction enzymes that generate incompatible termini and that the fragment of DNA to be cloned carries termini that are compatible with those of the doubly cleaved vector. - [Read Directional Cloning into Plasmid Vectors Protocol]

Category: DNA Protocols: DNA Extraction Protocols: DNA Extraction Polyacrylamide Gels

DNA Fragment Purification from Agarose or Acrylamide. The protocol for fragments from 200 bp to 10 kb the agarose purification is ideal.  For smaller fragments (20 bp to 400 bp) the acrylamide purification is preferred. - [Read DNA Fragment Purification from Agarose or Acrylamide]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols

Protocol for DNA fragment purification from agarose or acrylamide. For fragments from 200 bp to 10 kb the agarose purification is ideal.  For smaller fragments (20 bp to 400 bp) the acrylamide purification is preferred. - [Read DNA Fragment Purification from Agarose or Acrylamide Protocol]

Category: DNA Protocols: DNA Extraction Protocols: Agarose Gel DNA Extraction Protocols

DNA Fragment Purification from Agarose Protocol. This protocol is best for fragments from 200 bp to 10 kb the agarose purification is ideal. For smaller fragments (20 bp to 400 bp) the acrylamide purification is preferred. - [Read DNA Fragment Purification from Agarose Protocol]

Category: Chromatography Protocols: Column Chromatography Protocols

Fragment purification on Sephacryl S-500 spin columns , Bruce A. Roe. - [Read Fragment purification on Sephacryl S-500 spin columns]

Category: DNA Protocols: cDNA Library Protocols: cDNA Library Construction Protocols

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. - [Read Generation of a Library of Randomly Overlapping DNA Inserts Protocol]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Polyacrylamide Gels Protocols

The "crush and soak" method, which works best for DNAs <1 kb in size, can be used to recover both singleand double-stranded DNAs from polyacrylamide gels.  The yield of eluted DNA varies from <30% to >90% depending on the size of the DNA fragment. - [Read Isolation of DNA Fragments from Polyacrylamide Gels by the Crush and Soak Method Protocol]

Category: DNA Protein Interactions Protocols: Dnase Footprinting Assay Protocols

Dnase I is used to fragment a radiolabeled target DNA in the presence and absence of a nuclear extract.  A "footprint" is generated when a protein binds to the target and protects a specific segment of DNA from the nucleolytic activity of Dnase I. By comparing the electrophoretic mobility of the Dnase I cleavage products to those of a sequence ladder derived from the same DNA fragment, the position(s) of the DNA sequences recognized by DNA-binding proteins can be determined. - [Read Mapping Protein-binding Sites on DNA by Dnase I Footprinting 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: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Many replacement vectors (e.g., the EMBL series, {lambda}2001, and {lambda}DASH) contain a series of restriction sites, arranged in opposite orientations, at each end of the central stuffer fragment. Digestion of these vectors with two different restriction enzymes yields left and right arms, a stuffer fragment, and short segments of the polycloning sites. These can easily be removed from the arms by differential precipitation with isopropanol or spun-column chromatography. - [Read Preparation of Bacteriophage lambda DNA Cleaved with Two Restriction Enzymes Protocol]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

In this procedure, synthesis of cDNA is carried out in the presence of saturating concentrations of all four dNTPs and trace amounts of a single radiolabeled dNTP.  After subtraction hybridization, the enriched single-stranded cDNA is radiolabeled to high specific activity in a second synthetic reaction by extension of random oligonucleotide primers using the Klenow fragment of E. coli DNA polymerase. - [Read Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension Protocol]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Agarose Gels Protocols

A fragment of gel containing a band of DNA is excised and digested with agarase, which hydrolyzes the polymer to disaccharide subunits.  The released DNA is then purified by phenol extraction and ethanol precipitation.  The method works well for DNAs ranging in size from <5 kb to >20 kb. - [Read Recovery of DNA from Low-melting-temperature Agarose Gels: Enzymatic Digestion with Agarase Protocol]

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

Restriction Enzyme Troubleshooting Guide. Fermentas. Problems: Undigested or incompletely digested DNA, additional bands atypical, Diffused DNA zones, Low fragment ligation efficiency. - [Read Restriction Enzyme Troubleshooting Guide]

Category: Cytogenetics Protocols: RNA In Situ Hybridization Protocols

Protocol for RNA labeling by in vitro transcription of DNA with DIG, Biotin or Fluorescein RNA Labeling Mix. A PCR fragment that has the appropriate promoter ligated to its 5’-ends can also serve as a transcription template. The procedure described incorporates one modified nucleotide (DIG-, Biotin-, or Fluorescein-UTP) at approximately every 20 – 25th position in the transcripts. - [Read RNA Labeling by In Vitro Transcription of DNA with DIG, Biotin or Fluorescein RNA Labeling Mix]

Category: Mouse Protocols: Mouse Genetics Protocols

Protocol describes a simple procedure for DNA fragment isolation and purification. The resulting DNA fragment can then. be used for microinjection to produce transgenic mice - [Read Simple, Reliable Steps for DNA Fragment Isolation and Purification Protocol]