efficient Protocols

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture

Efficient derivation of mESCs. Bryja et al., 2005 Stem Cells Express. - [Read An efficient method for the derivation of mouse embryonic stem cells]

Category: Drosophila Protocols: Drosophila Genetics Protocols

Protocol described here produces chromatin with regularly spaced nucleosomes having physiological nucleosome repeat lengths; something that can be difficult to achieve with purified components. In addition, chromatin assembled with the S-190 Chromatin Assembly Extract contains the ATP-dependent chromatin remodeling factors necessary for efficient transcription. - [Read Chromatin Assembly on Template DNA with Transcription Factors and Drosophila S-190 Chromatin Assembl]

Category: Plant Biology Protocols

Protocol can be used for clearing intact non-ovule materials of arabidopsis, which can then be observed under Normarski optics. This is an efficient way to analyse root, seedling even flower development without sectioning. This protocol could also be used for clearing GUS stained material, after chlorophyll is removed by 70% ethanol. - [Read Clearing Arabidopsis Non-Ovule Materials With the HCG Solution Protocol]

Category: Plant Biology Protocols

Protocol can be used for clearing intact seeds of arabidopsis, which can then be observed under Normarski optics. This is an efficient way to analyse embryo and endosperm development without sectioning. - [Read Clearing Arabidopsis Ovule Materials with the Hoyer's Solution Protocol]

Category: Xenopus Protocols: Xenopus Injection Protocols

Differences in injection of X. laevis and X. tropicalis. Includes: X. tropicalis lays eggs about 4 hours after a boost of hCG; In vitro fertilization is not nearly as efficient in trops compared to laevis; X. tropicalis embryos are much softer than X. laevis embryos; X. tropicalis embryos whose jelly coats are removed by cysteine have a loose sticky vitelline membrane; X. tropicalis do not have a "summer slump". - [Read Differences in Injection of X. laevis and X. tropicalis]

Category: Cell Biology and Cell Culture: Cell Electroporation Protocols

Electroporation of Cell Lines With DNA. Electroporation for the efficient transfection of mammalian cells with DNA. Chu et al. Cellular Immunology Oxford - [Read Electroporation of Cell Lines With DNA]

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: PCR Protocols: cDNA Synthesis Protocols

This method of first strand cDNA synthesis is more efficient than Ready-to-Go beads.  You can use as little as 100ng total RNA and still detect you gene expression in PCR. - [Read First strand cDNA synthesis with Super Script II Protocol]

Category: Gene Delivery Protocols

This highly efficient in vivo gene transduction technique for laboratory mice. Hepatocytes are most effectively transduced by tail vein injection of a large volume of DNA solution in a short time. Practice with the injection technique is necessary!!! - [Read Hydrodynamics-Based Gene Transduction Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Isolation from Mammalian Cells Protocols

Procedure is used to prepare DNA simultaneously from many different types of samples or tissues.  Although the DNA is generally too small (approx.  80 kb) for efficient construction of genomic DNA libraries, it gives excellent results in Southern hybridizations and PCRs.  Cultured aneuploid mammalian cells (2 x 107, e.g., HeLa cells) yield 100 µg of DNA in a volume of 1 ml. - [Read Isolation of DNA from Mammalian Cells by Spooling Protocol]

Category: Plant Biology Protocols: Plant Genetics Protocols

The preparation of expressional cDNA libraries for use in the yeast two-hybrid system is quick and efficient when using the dedicated Clontech™ product, the MATCHMAKER Library Construction and Screening Kit 3. This kit employs SMART technology for the amplification of full-length cDNAs, in combination with cloning using homologous recombination. - [Read Isolation of Plant Transcription Factors Using a Modified Yeast One-Hybrid System]

Category: C. elegans Protocols: C. elegans Genetics Protocols

Protocol describes an efficient approach to isolate RNA from Caenorhabditis elegans. - [Read Isolation of RNA from C. elegans Protocol]

Category: Cloning Protocols

Protocol for ligating plasmid and target DNAs in low-melting-temperature agarose. Ligation in low-melting-temperature agarose is much less efficient than ligation with purified DNA in free solution and requires a large amount of DNA ligase. The method is used chiefly for rapid subcloning of segments of DNA in dephosphorylated vectors and assembling recombinant constructs. - [Read Ligating Plasmid and Target DNAs in Low-melting-temperature Agarose Protocol]

Category: PCR Protocols: Quantitative PCR Protocols

General guidelines for long-PCR conditions and enzyme mixtures. Efficient long-PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration. Includes: For PCR with low-complexity templates (e.g., plasmid and cosmid inserts); For PCR with moderate-complexity templates (e.g., bacterial genomic DNA); For PCR with high-complexity templates (e.g., human genomic DNA). - [Read Long-PCR Reagents and Guidelines]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Cell Sorting Protocols

This protocol has been established to maintain the efficient use of the AutoMACS cell sorter during periods of heavy use (e.g., three preparations of splenocytes from 16 spleens per week). Includes: Running the SAFE Clean Program; Clearing Debris. - [Read Maintenance of the AutoMACS Cell Sorter Protocol]

Category: Developmental Biology: Utilizing Model Organisms Protocols

Nonviral, DNA-mediated gene transfer is an alternative to viral delivery systems for expressing new genes in cells and tissues. The Sleeping Beauty (SB) transposon system combines the advantages of viruses and naked DNA molecules for gene therapy purposes; however, efficacious delivery of DNA molecules to animal tissues can still be problematic. Here we describe the hydrodynamic delivery procedure for the SB transposon system that allows efficient delivery to the liver in the mouse. - [Read Preferential Delivery of the Sleeping Beauty transposon System to Livers of Mice by Hydrodynamic i]

Category: Drosophila Protocols: Drosophila Genetics Protocols

Protocol describes how to produce a soluble nuclear extract rich in basal pol II transcription factors from Drosophila embryos. This is a cell-free extract that contains all the necessary transcription factors and is capable of accurate initiation of transcription by RNA polymerase II but is deficient in core histones and histone H1. - [Read Preparation of a Highly Efficient Transcription Extract from Drosophila Embryos Protocol]

Category: RNAi Technology Protocols: RNAi Drosophila Protocols

siRNAs produced upon the addition of dsRNA to Drosophila embryo extract are enriched in a micrococcus-nuclease-resistant fraction.  After proteinase K treatment and dephosphorylation with calf intestinal phosphatase, these siRNAs mediate efficient RNAi in vitro. - [Read Preparation of siRNAs from Drosophila Embryo Extracts Protocol]

Category: Cell Organelle Protocols: Nucleus Protocols

A simplified and efficient protocol for nonradioactive in situ hybridization to polytene chromosomes with a DIG-labeled DNA probe. Includes: Labeling the hybridization probe; Preparation and denaturation of polytene chromosomes from Drosophila, Chironomus, or other species; Hybridization and detection; - [Read Protocol for Nonradioactive In Situ Hybridization to Polytene Chromosomes With DIG-labeled DNA Prob]

Category: Nucleic Acid Purification Protocols

Protocol describes the standard method for nucleic acid purification by extraction first with phenol:chloroform (optionally containing hydroxyquiniline at 0.1%) and then with chloroform to remove any remaining phenol.  The procedure takes advantage of the fact that deproteinization is more efficient when two different organic solvents are used instead of one. - [Read Purification of Nucleic Acids by Extraction with Phenol:Chloroform Protocol]

Category: RNA Protocols: cDNA Libraries Library

Hoskins et al. 2005 NAR. "... describe a PCR-based method for directed screening of plasmid cDNA libraries. " - [Read Rapid and efficient cDNA library screening by self-ligation of inverse PCR products (SLIP)]

Category: Cloning Protocols: Mutagenesis Protocols

Protocol for rapid and efficient site-directed mutagenesis by the single-tube megaprimer PCR method. - [Read Rapid and Efficient Site-directed Mutagenesis by the Single-tube Megaprimer PCR Method Protocol]

Category: Western Blot Protocols: Immunoblot Stains for Total Protein Protocols

Protocol used to determine the position of molecular-weight markers and to ensure that efficient transfer of proteins to the blot has occurred, the total composition of the proteins can be determined by staining the membrane with Ponceau S (Staining Immunoblots for Total Protein Using Ponceau S) or India ink. - [Read Staining Immunoblots for Total Protein Using India Ink Protocol]

Category: Western Blot Protocols: Immunoblot Stains for Total Protein Protocols

Protocol used to determine the position of molecular-weight markers and to ensure that efficient transfer of proteins to the blot has occurred, the total composition of the proteins can be determined by staining the membrane with Ponceau S . - [Read Staining Immunoblots for Total Protein Using Ponceau S Protocol]

Category: Transfection Protocols: Calcium Phosphate Transfection Protocols

This protocol describes transfection of plasmid DNA into primary hippocampal neurons using DNA/calcium-phosphate (CaPO4) coprecipitation. The precise pH of the transfection medium and the incubation time of cells with the coprecipitate are critical for reproducible and efficient transfection. Once these parameters are optimized for a given plasmid, the method is easily adapted for transfection of other established cell lines. - [Read Transfection of Hippocampal Neurons with Plasmid DNA Using Calcium Phosphate Coprecipitation]