long Protocols

Category: Laboratory Model Organisms Protocols

Protocol describes the preparation of stocks of marine hypotrichs for long-term storage. Marine euplotids are maintained by serial passage of tube stocks. - [Read Maintenance of Stocks of Marine Hypotrichs Protocol]

Category: Cell Culture Protocols: Cell Culture Contamination Prevention and Removal Protocols

For both biological and economical reasons, it is important to eliminate mycoplasmas from cell cultures being used for basic research, diagnosis, and biotechnological production. The most commonly used method for elimination, inactivation, or suppression of mycoplasmas in cell cultures is treatment with antibiotics. In general, antibiotic therapies do not result in long-lasting, successful elimination. Also, the cytotoxic properties of antibiotics can cause undesirable side effects on cells. - [Read Mycoplasma Elimination Reagent Protocol]

Category: Stem Cell Protocols: Stem Cell Culture Protocols

Describes the steps in detail to isolate and expand neural stem cells in the form of neurospheres from tissue dissections of the post-natal mouse brain. Procedures for the long term passage of neurospheres and the cryopreservation of neurospheres are also provided. In addition to the guidelines and tips for generating neurosphere cultures, we describe the method to prepare neurospheres for analysis by light microscopy. - [Read Neural Stem Cell Culture: Neurosphere Generation, Microscopical Analysis and Cryopreservation]

Category: PCR Protocols

PCR Amplification Manual Roche. Guide to Standard PCR Protocl, Long PCR Protocol, Hot Start PCR, PCR and Cloning, Basic PCR, other. Roche Applied Science - [Read PCR Amplification Manual Roche]

Category: E Coli Protocols: E coli Genetics Protocols

Protocol for precision engineering of plant gene loci by homologous recombination cloning in Escherichia coli. Describe the basis for homologous recombination cloning in E. coli, the available tools and resources, together with a protocol for long range cloning and manipulation of an Arabidopsis thaliana gene locus, to create constructs co-ordinately driven by locus-specific regulatory elements. - [Read Precision Engineering of Plant Gene Loci by Homologous Recombination Cloning in E coli Protocol]

Category: Plant Biology Protocols: Plant Genetics Protocols

Describe the basis for homologous recombination cloning in E. coli, the available tools and resources, together with a protocol for long range cloning and manipulation of an Arabidopsis thaliana gene locus, to create constructs co-ordinately driven by locus-specific regulatory elements. - [Read Precision Engineering of Plant Gene Loci by Homologous Recombination Cloning in Escherichia Coli]

Category: Chromatography Protocols: Affinity Chromatography Protocols

Protocol describes the coupling of peptides or proteins to Affi-Gel 10 (Bio-Rad).  Affi-Gel 10 is a cross-linked agarose derivatized with N-hydroxysuccinimide (NHS) ester on a 10-atom-long spacer arm.  The NHS ester allows spontaneous coupling of proteins via free amino groups. - [Read Protein Ligand Affinity Chromatography Protocol]

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

Protocol for pulsed-field gel electrophoresis via contour-clamped homogeneous electric field gels. In CHEF gels, the electric field is generated from multiple electrodes, arranged in a square of hexagonal contour around the horizontal gel and clamped to predetermined potentials.  Using a combination of low field strengths, low concentrations of aragose, long switching intervals, and extended periods of electrophoresis, DNAs up to 5000 kb can be resolved. - [Read Pulsed-field Gel Electrophoresis via Contour-clamped Homogeneous Electric Field Gels Protocol]

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols: Long PCR Protocols

Long PCR Buffer, Cycle times and temperatures, Picking Primers, Hot Start Polymerases for Long PCR. Harvard-Lipper Center for Computational Genetics. Modified from Cheng et al. - [Read Reagents and Guidelines for Long PCR]

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]

Category: Plant Biology Protocols: Arabidopsis Transformation Protocols

Protocol for a simplified Arabidopsis transformation. Found that the MS salts, hormone, etc. make no difference, that OD of bacteria doesn't make much of a difference, that vacuum doesn't even make much of a difference as long as you have a decent amount of surfactant present. Plant health is still a major factor - healthy fecund plants make a big difference! With this method you should be able to achieve transformation rates above 1%. - [Read Simple Arabidopsis Transformation Protocol]

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols: Standard PCR Protocol

Standard Long PCR. This is a standard PCR protocol used on all first pass (unoptimized) PCR amplifications. UW-FHCRC - [Read Standard Long PCR]

Category: PCR Protocols: Standard PCR Protocol

Standard Long PCR. This is a standard PCR protocol used on all first pass (unoptimized) PCR amplifications. UW-FHCRC - [Read Standard Long PCR]

Category: Antibody Protocols: Antibody Handling Protocols

This protocol describes the methods of storage for antibody-containing sera. Antibodies are resistant to a broad range of mildly denaturing conditions, so long-term storage is relatively easy. - [Read Storage of Sera Protocol]

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols: Long PCR Protocols

Succesful Long PCR Amplification Conditions Guide. Takara Bio. - [Read Succesful Long PCR Amplification Conditions Guide]

Category: RNAi Technology Protocols: RNAi Drosophila Protocols

Protocol describes how to prepare double-stranded RNA (dsRNA) for RNA interference in Drosophila by synthesis of individual RNA strands from linearized plasmid templates, followed by annealing of the strands.  DsRNA molecules with a length of 500-800 bp seem to be most active.  The dsRNA can be made from cDNA or genomic DNA templates, as long as most of the dsRNA corresponds to presumptive exon sequence. - [Read Synthesis of dsRNA for RNAi in Drosophila: Plasmid Template Method Protocol]

Category: Transfection Protocols: Calcium Phosphate Transfection Protocols

This protocol describes two transfection methods for expressing GFP-tagged actin in primary neurons. The lipid reagent DOTAP (Roche Diagnostics) method produces actin-GFP-expressing hippocampal neurons that survive well during long periods in culture. The calcium phosphate method can be used to transfect neurons that have already been growing on coverslips in vitro. Transfected cells suitable for imaging can be obtained in cultures up to 15 days in vitro. - [Read Transfecting Cultured Hippocampal Neurons with an Actin-GFP Plasmid]

Category: Neuroscience Protocols: Neuronal Cell Culture Protocols

Protocol describes two transfection methods for expressing GFP-tagged actin in primary neurons.  The lipid reagent DOTAP (Roche Diagnostics) method produces actin-GFP-expressing hippocampal neurons that survive well during long periods in culture. - [Read Transfecting Cultured Hippocampal Neurons with an Actin-GFP Plasmid Protocol]

Category: PCR Protocols: Long PCR Protocol. Extended PCR.

Troubleshooting Long PCR Roche - [Read Troubleshooting Long PCR Roche]