clone Protocols

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Bacterial clone DNA Protocol for FISH. - [Read Bacterial clone DNA Protocol for FISH]

Category: Cell Biology and Cell Culture: Cell Transfection Protocol

Protocol for cell transfection, clone screening and picking cell clones. Bjorkman Group. California I.T - [Read Calcium phosphate Tranfection for MDCK cells]

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

Protocol for cloning genes from a phage library. Includes: Titer and plate out phage; Lift plaques onto filters and prepare them for screening; Make a probe; Hybridize the probe to the filters; Wash the filters and expose to film; Purify putative plaques; Excise plasmid from the desired phage. - [Read Clone Genes From a Phage Library Protocol]

Category: Drosophila Protocols: Drosophila Culture Media Protocols

Protocol for Drosophila growth conditions. Includes: MAINTENANCE; Semi-adherent cell lines; Adherent cell lines; SL2, S2C, S2*; DL2, DL1, S2R+, Kc167, Clone 8, S3; BG2. - [Read Drosophila Growth Conditions 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: Cloning Protocols

Protocol describes a method for DNA fragmentation by sonication. During sonication, DNA samples are subjected to hydrodynamic shearing by exposure to brief periods of sonication. DNA that has been sonicated for excessive periods of time is extremely difficult to clone. - [Read Fragmentation of DNA by Sonication Protocol]

Category: E Coli Protocols: E coli Genetics Protocols

Protocol for the generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system. Technology requires flanking DNA to be cloned into a temperature sensitive vector but the resulting clone allows great flexibility for further modification of the target sequence. It is therefore highly suited to the study of genes in which several rounds of changes are envisaged. - [Read Generation of Gene Deletions and Gene Replacements in Escherichia coli Protocol]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

There are essentially three parts to this protocol: 1. growth of at least 5x10e8 pfu phage to provide an inoculum growth of a larger liquid lysate that will produce about 5x10e12 pfu; 2. concentration and purification of the phage, and; 3. DNA preparation. - [Read Growth and Purification of 25-100 ug Lambda Clone DNA Protocol]

Category: PCR Protocols

PCR polymerase costs can be high. If you are willing to work, you can produce bacteria containing the clone. It appears to produce lots of Taq and is quite stable. The proceedure takes 4 days start to (15 000 units of Taq) finish. The Taq also appears ver - [Read Home-made Taq Polymerase Purification]

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: Bacteria Genetics Protocols

Protocol for the identification of positive GATEWAY expression clones when both the pENTRY and pDEST vectors contain the same marker for bacterial selection. Protocol describes ways in which difficult vector combinations can be used effectively to obtain the appropriate expression clone without having to convert the pENTRY clone or pDEST clone to vectors with compatible antibiotic resistances. - [Read Identification of Positive GATEWAY Expression Clones Protocol]

Category: PCR Protocols: Inverse PCR Protocols

Inverse PCR is used to amplify and clone unknown DNA that flanks one end of a known DNA sequence and for which no primers are available.  The technique involves digestion by a restriction enzyme of a preparation of DNA containing the known sequence and its flanking region.  The individual restriction fragments (many thousands in the case of total mammalian genomic DNA) are converted into circles by intramolecular ligation, and the circularized DNA is then used as a template in the PCR. - [Read Inverse PCR Protocol II]

Category: Cloning Protocols

Protocol can be used to optimize ligation conditions for difficult to clone (e.g. very large) fragments. The principle is to independently characterize the ligation kinetics of the vector and insert DNA fragments and then to combine them in optimal ratios. - [Read Ligation Optimization Protocol]

Category: DNA Microarray Protocols

This protocol describes clone handling, plate replication, and DNA template preparation in a 96 well format. Hasseman. TIGR Microarray Protocols - [Read MICROARRAY cDNA CLONE GROWTH AND TEMPLATE]

Category: PCR Protocols

PCR can be used to identify rare DNA sequences in DNA libraries by increasing the abundance of a particular sequence.  This is accomplished by subdividing the original library into pools of decreased complexity and screening each pool or group of pools for a given DNA sequence.  A pool that contains the desired clone is subsequently subdivided into smaller pools, each of which is screened using the same PCR protocol that was used for the primary screen. - [Read PCR-Based Screening of DNA Libraries Protocol]

Category: Immunology: T Cell Protocols

In the first protocol, IL-2-producing murine T cells are measured following stimulation by the mitogen Con A. The second protocol provides a modification for using human responder cells. The second protocol is used for estimating the proportion of cells that can generate a clone of cytotoxic effector cells when stimulated by Con A with the addition of IL-2. - [Read Quantitation of Functional T Cells by Limiting Dilution Protocols]

Category: PCR Protocols: cDNA Synthesis Protocols

3'-RACE reactions are used to isolate unknown 3' sequences or to map the 3' termini of mRNAs onto a gene sequence.  3'-RACE requires knowledge of a small region of sequence within either the target RNA or a partial clone of cDNA.  A population of mRNAs is transcribed into cDNA with an adaptor-primer consisting at its 3' end of a poly(T) tract and at its 5' end of an arbitrary sequence of 30-40 nucleotides. - [Read Rapid Amplification of 3' cDNA Ends 3'-RACE Protocol]

Category: PCR Protocols: cDNA Synthesis Protocols

This method is used to extend partial cDNA clones by amplifying the 5' sequences of the corresponding mRNAs.  The technique requires knowledge of a small region of sequence within the partial cDNA clone.  During PCR, the thermostable DNA polymerase is directed to the appropriate target RNA by a single primer derived from the region of known sequence. - [Read Rapid Amplification of 5' cDNA Ends 5'-RACE Protocol]

Category: Fungi Protocols: Aspergillus Protocols

Protocol for the transformation of Aspergillus niger. This procedure is done by first digesting the outer cell wall, forming protoplasts, and then by making holes in the membrane through which the dna can enter using calcium chloride and polyethylene glycol. Includes: Protocol for making A.niger protoplasts; Transformation; Plating. - [Read Transformation of Aspergillus niger Protocol]

Category: Transgenic Mouse Protocols

Clone the Transgene, transgene Screening, Transgene Expression, DNA microinjection, Identify Transgenic Founders, Transgenic Analysis. Transgenic Animal Web. - [Read Transgenic Outline: Mice and Rats]

Category: Plant Biology Protocols

Alfalfa is an outcrossing species, cultivars consist of populations rather than individual homozygous or inbred lines. After only two cycles of self-pollination, severe inbreeding depression eliminates selfed individuals from populations. To obtain sufficient plant material of relative genetically uniformity (these plants will still necessarily be heterozygous) for experimental purposes, it may be necessary to propagate a single individual or clone. - [Read Vegetative Propagation of Alfalfa by Stem Cuttings]

Category: Xenopus Protocols: Xenopus Genetics Protocols

Xenopus cDNA libraries. Includes: General cDNA clone providers. - [Read Xenopus cDNA Libraries]