PCR cycle steps, protocol using SuperScript II reverse transcriptase (Life Technologies) and Taq polymerase, Gene specific primer, T17 Adapter primer and an Adapter primer. Dr.Dawson, Nottingham. - [Read 3' Rapid Amplification of cDNA Ends (RACE) Protocol]
Information on how to quantitatively assess DNA content. Includes: Propidium iodide staining protocol and staining of DNA by Mithramicin Protocol - [Read Cell Cycle Analysis - Propidium Iodide]
protocol describes a method for the synchronization of cell populations using centrifugal elutriation. The method relies on the fact that cell size increases linearly as cells proceed through the cell cycle. Cells of similar size (and cell cycle phase) are eluted stepwise from the cell chamber, with the smallest size (those in early G1) being eluted first. Using this procedure, it is possible to obtain relatively pure populations of cells at various points in G1, S, and G2/M. - [Read Cell Synchronization Using Centrifugal Elutriation Protocol]
This protocol describes a method for the synchronization of cell populations using centrifugal elutriation. The method relies on the fact that cell size increases linearly as cells proceed through the cell cycle. Cells of similar size (and cell cycle phase) are eluted stepwise from the cell chamber, with the smallest size (those in early G1) being eluted first. Using this procedure, it is possible to obtain relatively pure populations of cells at various points in G1, S, and G2/M. - [Read Cell Synchronization Using Centrifugal Elutriation Protocol]
Describes assays used to determine the distribution of a population of cells to the different stages of the cell cycle as analyzed by flow cytometry. Staining the DNA with different fluorescent dyes, propidium iodide or DAPI, is one of the most direct ways of staging the cells based on DNA content. - [Read Determining Cell Cycle Stages by Flow Cytometry Protocol]
DNA Sequencing Methods- http://www.genome.ou.edu/protocol_book/protocol_partIV.html#IV.C
Methods for DNA sequencing. Includes: Bst-catalyzed radiolabeled DNA sequencing; Radiolabeled sequencing gel preparation, loading, and electrophoresis; Taq-polymerase catalyzed cycle sequencing using fluorescent-labeled dye primers; Taq-polymerase catalyzed cycle sequencing using fluorescent-labeled dye terminator reactions; Sequenase[TM] catalyzed sequencing with dye-labeled terminators; etc. - [Read DNA Sequencing Methods]
FIXATION and DNA Staining for Cell Cycle Analysis Protocol. This method of DNA staining utilizes ethanol to fix the cells and permeabilize the membrane, which allows the dye (Propidium Iodide) to enter the cells. Propidium Iodide (PI) is a DNA-binding fluorochrome that intercalates in the double-helix. Ribonuclease-A is used to eliminate the staining of double-stranded RNA. - [Read FIXATION and DNA Staining for Cell Cycle Analysis]
Analysis of Cell Cycle Based on DNA Content. FIXATION PROTOCOL FOR FLOW CYTOMETRIC ... Liberate cells from tissue culture flask by trypsin digestion and ... FIXATION PROTOCOL FOR FLOW CYTOMETRIC ANALYSIS OF DNA BY PI STAINING. Auburn University, College of Veterinary Medicine. You. - [Read FIXATION PROTOCOL FOR FLOW CYTOMETRIC ANALYSIS OF DNA BY PI STAINING]
Genotyping Ts65Dn mice is based on doing simultaneous quantitative PCR amplification of a gene or genes in the Ts65Dn chromosome and a control gene on another chromosome (in this case Apob) and comparing the average change (delta) in threshold cycle (CT) - [Read Genotyping Mice Using Real Time]
Specific molecular components can be efficiently labeled by a combination of three methods: chemical transfection of GFP-fusion constructs, staining of chromosomes with the DNA-specific, fluorescent dye Hoechst 33342, and microinjection of fluorescently conjugated proteins. This procedure provides an example of using all three methods in sequence to label components of living HeLa cells. These methods should be followed in the order presented, but any of them can be omitted when not needed. - [Read Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle]
Fluorescence microscopy provides a powerful tool for imaging molecular components in living cells. Specific molecular components can be efficiently labeled by a combination of three methods: chemical transfection of GFP-fusion constructs, staining of chromosomes with the DNA-specific, fluorescent dye Hoechst 33342, and microinjection of fluorescently conjugated proteins. This procedure provides an example of using all three methods in sequence to label components of living HeLa cells. - [Read Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle]
3' Rapid Amplification of cDNA Ends RACE Using PCR Protocol. This protocol contains the steps for 3' end rapid amplification of mRNA by PCR. The first-strand cDNA is synthesized from total or poly(A+) RNA by priming from the poly-A tail of the mRNA using a oligo (dT) adaptor primer. The cDNA is then amplified via PCR using a gene-specific primer and an adaptor primer.
This protocol describes the electroporation of the BMH 81-17 mut S strain that is recommended for tranformation of the site directed mutagenesis of dsDNA (See Protocol on Site-Directed Mutagenesis on Double Stranded DNA). BMH 81-17 mut S are a mismatch repair defective (mut S) Escherichia coli strain. The probability that the two mutations will cosegregate during the first round of DNA replication is increased in this strain.