insertion Protocols

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Colony PCR Protocol II - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4141

Protocol describes the use of PCR to screen for bacteria that carry recombinant plasmids. The PCR can be carried out using the same primers as for amplification of the cloned insert. To determine the orientation of the insert, a third, insert-specific primer that is asymmetrically distanced from the clonal insertion site can be used. - [Read Colony PCR Protocol II]

DNA Ligation Protocol from Takara Shuzo Co. - http://www.hawaii.edu/microbiology/MO/ligation.htm

Category: Molecular Biology Protocols: DNA Ligation Protocols

Insertion of DNA into Plasmid Vectors, Insertion of DNA into l Phage Vectors, Self-Circularization of Linear DNA (Intramolecular Ligation), Linker (Adaptor) Ligation. Alam's Lab Univ Hawaii. - [Read DNA Ligation Protocol from Takara Shuzo Co.]

Gene Delivery by Direct Injection (Microinjection) Using a Pulsed-Flow System Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/30/pdb.prot4653

Category: Microinjection Protocols: Microinjection of DNA Protocols

This protocol describes a method for pulsed-flow microinjection using the Eppendorf FemtoJet injector and Eppendorf InjectMan; this is the most common type of pulsed-flow microinjection system currently being used. The advantage of this type of system over a controlled-flow system is that much more control is available over the injection parameters, reducing variability in injections. In addition, the system allows a diagonal insertion of the needle into the cell. - [Read Gene Delivery by Direct Injection (Microinjection) Using a Pulsed-Flow System Protocol]

Handling and Using Insertion Libraries - http://ygac.med.yale.edu/mtn/protocol.stm

Category: Yeast Protocols

Handling and using insertion libraries. Includes: Curing strains of endogenous 2-micron; mTn-lacZ/LEU2 insertion library; mTn-3xHA/lacZ insertion library; mTn-3xHA/GFP insertion library. - [Read Handling and Using Insertion Libraries]

PCR-Mediated Gene Disruption: One-Step Method Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4169

Category: PCR Protocols

DNA prepared by PCR-mediated gene disruption can be used to transform yeast in gene replacement experiments. This protocol uses two primers, tailed with approximately 50 nucleotides homologous to the gene of interest, that target insertion of the PCR product to that locus. Each primer ends with a universal sequence that is designed to amplify various selectable markers from plasmid templates. - [Read PCR-Mediated Gene Disruption: One-Step Method Protocol]

Screening DNA Pools for T-DNA Insertions in Arabidopsis Genes Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/28/pdb.prot4622

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

A powerful way to identify a mutation in the gene of interest and to test mutant plants for phenotypes that are predicted to result from loss of function of that gene is by PCR screening. Pools of insertion lines are screened using one primer corresponding to the gene of interest and one primer corresponding to the end of the insertion element. The synthesis of a product indicates the presence of an insertion in the gene of interest. - [Read Screening DNA Pools for T-DNA Insertions in Arabidopsis Genes Protocol]

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Vector Design Protocol for Transgenic Mice Generation

The protocol gives general considerations for the design of targeting vectors for transgenic mice. The protocol shares tips in the design of knock-out and knock-in vectors and some of their strategies for producing homologously recombined embryonic stem cells.