series Protocols

Category: Immunology: Chemotaxis

96-well Chemotaxis Chambers Protocol. Neuroprobe. - [Read 96-well Chemotaxis Chambers Protocol]

Category: Molecular Biology Protocols: DNA Ligation Protocols

Addition of linkers/adaptors to blunt-end DNA fragments. Ligation of mixture containing DNA and linkers/adaptors. Powell Gannon. Oxford Practical Approach Series. - [Read Addition of linkers/adaptors to blunt-end DNA fragments PDF]

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

Aggregation of ES Cells with Mouse Morula-state Embryos. Oxford Practical Series Approach. - [Read Aggregation of ES Cells with Mouse Morula-state Embryos]

Category: Cell Biology and Cell Culture: Apoptosis Protocols

Caspase Detection Methods for Apoptosis. Zeuner et al. Purdue Cytometry CD-ROM Series. - [Read Caspase Detection Methods for Apoptosis]

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

Cloning Enzymes a Guide Promega. An Enzyme Resource Guide series, highlights those enzymes important in nucleic acid cloning procedures. Promega - [Read Cloning Enzymes a Guide Promega]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

In this protocol, sample and competitor RNAs are reverse transcribed (separately) in a pilot experiment.  A constant amount of sample RT product is then combined with a 2-logserial dilution of competitor RT product for PCR. Procedure provides an approximate copy number for the sample, which is then fine-tuned by repeating the experiment with a series of twofold dilutions of competitor.  The experiment includes controls for sample-to-sample variations in RT efficiency. - [Read Competitive RT-PCR: Estimation of Copy Number Protocol]

Category: Cell Biology and Cell Culture: Apoptosis Protocols

Detection of apoptotic process in situ using immunocytochemical and TUNEL assays. Barbieri et al. Purdue Cytometry CD-ROM Series - [Read Detection of apoptotic process in situ using immunocytochemical]

Category: Molecular Biology Protocols: DNA Ligation Protocols

DNA ligation Method, Equipment and reagents, T4 DNA ligase. Gannon and Powell. Oxford Practical Approach Series. - [Read DNA Ligation Protocol PDF]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Mitochondria Isolation Protocols

Generally in iodixanol gradients the density of organelles decreases in the series: peroxisomes, mitochondria, lysosomes, ER, Golgi, although in Dictyostelium discoideum, the lysosomes are denser than the mitochondria. Iodixanol gradients can usually provide satisfactory resolution of all these membrane particles although it may be necessary to modulate either the gradient or centrifugation parameters in order to optimize a particular separation. - [Read Fractionation of Mitochondria, Lysosomes, Peroxisomes, ER and Golgi in Pre-formed Iodixanol Gradient]

Category: Cell Culture Protocols: Cell Immortalization Protocols

Cultivating animal cells in the laboratory is an indispensable technique for cell biologists. However, most normal primary cell lines, while faithfully reproducing the phenotype of their tissue of origin, do not grow indefinitely in culture. After a series of population doublings (the number of which varies by species, cell type, and culture conditions) primary cells enter a state where they no longer divide. - [Read Immortalization of Cells in Culture]

Category: Molecular Biology Protocols: DNA Ligation Protocols

10 × tailing buffer. Tdt procedure. Terminal transferase and cloning. Powell and Gannon. Oxford Practical Approach Series. - [Read Ligation of DNA fragments by homopolymer tailing]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes the acquisition and processing of confocal fluorescent images of live cells expressing yellow fluorescent protein (YFP), with a spinning disk confocal head on a Zeiss Axiovert 200 M microscope. Protocol includes: Description of Microscope and Imaging Setup; Description of Acquisition Parameters; Movie Processing. - [Read Live Cell Spinning Disk Confocal Fluorescence Imaging of Cells- YFP Time Series for Markers]

Category: Bacterial Cell Culture Protocols: Bacterial Stocks Protocols

Plasmid (pUC series) containing genomic DNA fragments are maintained in E. coli strain DH5aTM. The E. coli cultures are routinely cultured at 37 C on Luria-Bertani (LB) agar on or in LB broth containing Ampicillin (30 µg/ml) or Carbenicillin (50 µg/ml broth, 100 µg/ml agar). E. coli strains are usually preserved in stab agar or glycerol for mid-term storage and lyophilized for long-term storage. - [Read Maintenance of Probes in Bacteria Including Escherichia coli Protocol]

Category: DNA Microarray Protocols

Making a scanning array: in situ synthesis of oligonucleotides on a solid substrate. Elder et al., Oxford Practical Approach Series. - [Read Making a scanning array: in situ synthesis of oligonucleotides on a solid substrate]

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

Protocol fopr markers of pulsed-field gel electrophoresis. Markers for pulsed-field gel electrophorsis can be generated by ligation of linear monomers of bacteriophage {lambda} DNA (48.5 kb) into a nested series of concatemers.  This procedure yields a series of concatemers that contain up to 20 tandemly arranged copies of bacteriophage DNA. - [Read Markers for Pulsed-field Gel Electrophoresis Protocol]

Category: Plant Biology Protocols: Photosynthesis and Respiration Protocols

The physiological reactions of mitochondria and chloroplasts can be reduced to a series of electron transfers, catalyzed by specific enzymes found within the organelles. Thus, we can study the component processes of photosynthesis and respiration by isolating the organelles and measuring specific enzyme activity associated with that organelle. - [Read Photosynthesis and Respiration - Introduction]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Bacteriophage {lambda} vectors that allow genetic selection of recombinant bacteriophages (e.g., the EMBL series, {lambda}2001, {lambda}DASH, {lambda}ZAP, and {lambda}gt10) can be prepared for cloning by simple digestion by one or more restriction enzymes. - [Read Preparation of Bacteriophage lambda DNA Cleaved with a Single Restriction Enzyme Protocol]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Many replacement vectors (e.g., the EMBL series, {lambda}2001, and {lambda}DASH) contain a series of restriction sites, arranged in opposite orientations, at each end of the central stuffer fragment. Digestion of these vectors with two different restriction enzymes yields left and right arms, a stuffer fragment, and short segments of the polycloning sites. These can easily be removed from the arms by differential precipitation with isopropanol or spun-column chromatography. - [Read Preparation of Bacteriophage lambda DNA Cleaved with Two Restriction Enzymes Protocol]

Category: PCR Protocols: Quantitative PCR Protocols

Quantitative PCR involves co-amplification of two templates: a constant amount of a preparation containing the desired target sequence and serial dilutions of a reference template that is added in known amounts to a series of amplification reactions.  The concentration of the target sequence is determined by simple interpolation into a standard curve. - [Read Quantitative PCR Protocol II]

Category: Molecular Biology Protocols: DNA Ligation Protocols

Powell and Gannon. Oxford Practical Apporach Series. - [Read Removal of Excess DNA Linkers / Adaptors]

Category: DNA Protocols: cDNA Library Protocols

A cDNA library constructed in a plasmid expression vector of the pUC, pUR, or pEX series is plated on agar medium and then replicated onto filters, which are transferred to plates containing IPTG. After 2-4 hours of induction, the colonies are lysed with chloroform and then screened with appropriate antibodies. - [Read Screening Expression Libraries Constructed in Plasmid Vectors Protocol]

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

Sequential digestion of DNA using two restriction endonucleases. Gannon and Powell. Oxford Practical Approach Series. - [Read Sequential digestion of DNA using two restriction endonucleases]

Category: PCR Protocols: Colony PCR

The following colony PCR protocol has been designed to be performed in individual reaction tubes. We usually test three colonies from each transformation along with a wild-type control. A series of five different PCR tests are performed on each colony to - [Read Single tube confirmation PCR protocol]

Category: DNA Microarray Protocols

Reusing DNA Microarrays using Stripping. Elder et al Oxford Practical Series. - [Read Stripping DNA Microarray array for re-use reusing]

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols

TAIL PCR Protocol. TAIL is a series of reactions that are intended to map where a T-DNA (transfer DNA) has inserted within the genome. The main components of the 3 reactions are the AD (Arbitrary Degenerate) primers, border primers, and DNA from the T-DNA - [Read TAIL PCR Protocol]