sequences Protocols

Category: Bioinformatics

Using AFPredictor, it was demonstrated that ‘ordered surface carbons’ (OSCs) are a distinguishing feature of AFPs and, more specifically, their ice-binding surfaces. AFPredictor identified AFPs from within a large set of structures with greater than 99% specificity. Furthermore, it was used to identify a novel ice-binding protein by screening a library of homology modeled structures based on cDNA sequences obtained from cold-acclimated winter rye (Secale cereale). - [Read A Computational Screening protocol for Antifreeze/Ice-Structuring Proteins]

Category: DNA Protocols: Cosmid Library Protocols

Amplification of cosmid libraries may result in distorted representation of cloned genomic sequences and should be avoided wherever possible. If amplification should become necessary, it is best to expand the library by growth in liquid medium. - [Read Amplification and Storage of a Cosmid Library: Amplification in Liquid Culture Protocol]

Category: DNA Protocols: Cosmid Library Protocols

Amplification of cosmid libraries may result in distorted representation of cloned genomic sequences and should be avoided wherever possible. In this method of amplification, distortion of the library is rarely a problem because at no stage are bacteria containing different recombinant cosmids grown in competition with one another. - [Read Amplification and Storage of a Cosmid Library: Amplification on Filters 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: DNA Protocols: DNA Extraction Protocols: Extracting Ancient DNA

The DNA obtained was invariably of a low average molecular size and damaged by oxidative processes, but PCR can be used to amplify and study short mitochondrial DNA sequences thatare of anthropological and evolutionary significance. SVANTE PAABO, PNAS. - [Read Ancient DNA: Extraction, characterization, molecular cloning, and enzymatic amplification. PDF]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Protocol for combined DNA in situ hybridization and immunocytochemistry for the simultaneous detection of nucleic acid sequences, proteins, and incorporated BrdU in cell preparations. Includes: Cell preparations and BrdU labeling; Detection of antigen by immunocytochemistry (ICC); Visualization of ICC antigen; -Gal-BCIG reaction (for producing a blue precipitate visible under brightfield microscopy); Cell processing for in situ hybridization; In situ hybridization (ISH); etc... - [Read Combined DNA In Situ Hybridization and Immunocytochemistry Protocol]

Category: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Protocol describes a high sensitivity indirect detection procedure for DIG-labeled hybridization probes.  The procedure uses the components of the HNPP Fluorescent Detection Set to form a fluorescent precipitate of HNPP (2-hydroxy-3-naphthoic acid-2’-phenylanilide phosphate) and Fast Red TR at the site of hybridization.  This procedure can be used to detect single copy sequences as small as 1 kb on human metaphase chromosomes. 
    - [Read DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System Protocol]

Category: Plant Biology Protocols: Plant Genetics Protocols

Microsatellite markers, also referred to as STMS (SequenceTagged Microsatellite Sites) or STR (Short Tandem Repeats) are widely used as molecular markers for intraspecific genotyping, molecular mapping and breeding purposes. The method described is an efficient,fast and relatively inexpensive way to obtain microsatellite markers without post-cloning selection methods. So far, the method has been successful in onion (Allium cepa L.), a plant with a large genome and for pathogenic fungi. - [Read Enrichment for Microsatellite Sequences in Onion (Allium cepa L.) Protocol]

Category: E Coli Protocols: E coli Protein Protocols

Protocol for the expression of cloned genes in E. coli using IPTG-inducible promoters. Protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying IPTG-inducible promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins. - [Read Expression of Cloned Genes in E. coli Using IPTG-inducible Promoters Protocol]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying bacteriophage {lambda} pL promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins. - [Read Expression of Cloned Genes in E. coli Using the Bacteriophage lambda pL Promoter Protocol]

Category: E Coli Protocols: E coli Protein Protocols

Protocol for expression of cloned genes in E. coli using the bacteriophage lambda pL promoter. Protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying bacteriophage lambda pL promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins. - [Read Expression of Cloned Genes in E. coli Using the Bacteriophage lambda pL Promoter Protocol]

Category: E Coli Protocols: E coli Protein Protocols

Protocol for the expression of cloned genes in E. coli using the bacteriophage T7 promoter. Protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying bacteriophage T7 promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins. - [Read Expression of Cloned Genes in E. coli Using the Bacteriophage T7 Promoter Protocol]

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Method assesses cellular mRNA transcripts in tissue sections and cell cultures using unique short anti-sense primers directed against sequences in particular protein(s). The unlabeled synthetic cDNA oligonucleotide primers are extended complementary to a sense mRNA transcript using reverse transcriptase and labeled through incorporation of a fluorescent-labeled dUTP nucleotide base. This procedure provides rapid detection of low abundance mRNA messages that can be related to other cellular.... - [Read Fluorescent In Situ Transcription in Cells and Tissues Protocol]

Category: Yeast Protocols: Yeast Protein Protocols: Yeast Protein Protein Interactions Protocols

This protocol describes the first step in constructing an array: amplification of the predicted ORFs that are to be included in the array. Gene-specific primers containing vector-specific flanking sequences that facilitate recombinational cloning are used to amplify each ORF. A secondary amplification can be used to extend the length of the homologous vector sequence flanking the ORF. - [Read Genome-Wide Analysis of Protein-Protein Interactions Using a Two-Hybrid Array: Amplification of ORFs]

Category: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast PCR Protocols

To identify the YAC subclones containing both a human insert and a portion of either the left or right arm of the pYAC4 vector. Identification of these clones is necessary in order to do YAC chromosome walking, and is also useful in the determination of whether a particular YAC clone has a contiguous human insert or whether a co-cloning event has occurred. Vector arm sequences are identified using pBR322 fragments from a BamHI-PvuII double digest. - [Read Identification of End Clones in YAC Subclone Libraries Protocol]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Protocol describes how to identify cloned cDNAs encoding proteins that bind to specific DNA sequences. The methods used are very similar to those used for immunological screening of expression libraries except that the nitrocellulose filters carrying immobilized proteins are screened with 32P-labeled double-stranded DNA rather than with antibodies. - [Read Identifying DNA-binding Proteins in Bacteriophage ambda Expression Libraries Protocol]

Category: Cytogenetics Protocols

In situ hybridisation to alpha satellite sequences (chromosome specific). Method book .net - [Read In situ hybridisation to alpha satellite sequences (chromosome specific)]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

In situ hybridisation to alpha satellite sequences (chromosome specific) protocol. - [Read In Situ Hybridisation to Alpha Satellite Sequences (Chromosome Specific) Protocol]

Category: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Protocol for in situ hybridisation to alpha satellite sequences (chromosome specific). - [Read In Situ Hybridisation to Alpha Satellite Sequences Protocol]

Category: RNA Protocols: In Vitro Transcription T7 SP6 T3 Protocols

RNA, in vitro synthesis using T3 T7 promoters. Dr. Dawson Univ. Nottingham. - [Read In vitro RNA synthesis from plasmid-borne sequences under the control of T phage promoters]

Category: PCR Protocols: PCR Cloning Protocols

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. - [Read Isolating the Ends of Genomic DNA Fragments Cloned in High-capacity Vectors]

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: PCR Protocols: cDNA Synthesis Protocols

In MOPAC, the amino-terminal and carboxy-terminal sequences of a peptide are used to design two redundant families of oligonucleotides encoding the aminoand carboxy-terminal sequences of the peptide.  The primers are used either to amplify a segment of cDNA prepared by RT-PCR from a tissue known to express the protein or to amplify a segment of DNA from an established genomic or cDNA library. - [Read Mixed Oligonucleotide-primed Amplification of cDNA MOPAC Protocol]

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

Primer pairs will amplify sequences present as a single copy in the mouse genome with the Universal Genotyping Protocol. Includes: b-Galactosidase (LacZ); cre-recombinase; CFP; diphtheria toxin; dsRED; Fabpi-200; Fabpi-500; flp recombinase; GFP/BFP/YFP; human growth hormone (complete); human growth hormone (transcriptional stop); luciferase (click-beetle); luciferase (firefly); neomycin phosphotransferase; SRY (male-specific); tTA (tet-on). - [Read PCR Genotyping Primer Pairs Protocols]