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Equipment

1. Parafilm

• Petri dishes/100mm tissue culture dishes
• Marker pen
• Microscope slides
• Glass capillaries
• Very fine tweezers
• MBS solution*
• Plastic transfer pipettes

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General Comments

The microscopic tip of the glass microcapillary has an inner diameter between 0.2 and 1 µm . This capillary is back loaded with the substance to be transferred into the cells cultured for microinjection. Typical substances include purified antibodies, DNA, RNA, peptides, or oligonucleotides.

To visualize and evaluate the success of a microinjection experiment these substances are typically mixed with dyes or labeled with fluorescent markers such as flourescein or rhodamine. After the capillary has pierced the cell, a certain amount of transfer substance (approximately 10% of the cell volume) is transferred from the capillary into the cell due to pressure exerted on the capillary via the microinjector [2]. Because the diameter of the capillary is very small, particles in the injection solution can quickly result in blockage of the capillary. Therefore, exchange of the capillary is a common practice during microinjection experiments and the frequency of the capillary exchange depends on t

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Author: David W. Mount

Adapted from “Phylogenetic Prediction,” Chapter 7, in Bioinformatics: Sequence and Genome Analysis, 2nd edition, by David W. Mount. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2004.

INTRODUCTION

Three methods--maximum parsimony, distance, and maximum likelihood--are generally used to find the evolutionary tree or trees that best account for the observed variation in a group of sequences. Each of these methods uses a different type of analysis. Programs based on distance methods are commonly used in the molecular biology laboratory because they are straightforward and can be used with a large number of sequences. Maximum likelihood methods are more challenging and require a greater understanding of the evolutionary models on which they are based. Because they involve so many computational steps and because the number of steps increases dramatically with the number of sequences, maximum likelihood programs are limited to a smaller number of sequences. T

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Method:

1. Incubate sections in the incubating medium at 37°C for 5 - 12 hours. Long incubation periods are needed to get significantly visible reaction product.

• Wash in distilled water for 2 minutes.
• Counterstain with Methyl Green for 5 minutes.
• Wash in distilled water for 2 minutes.
• Air dry and coverslip.
• Results:
  • Nuclei - dark green
• Cytoplasm - light green

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Procedure:

1. Incubate sections in the incubating medium at room temperature for 1 - 3 hours. Two hours is sufficient in most cases.

• Wash in distilled water for 2 minutes.
• Counterstain with Nuclear Fast Red for 5 - 10 minutes.
• Wash in distilled water for 2 minutes.
• Air dry and coverslip.

Results:

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Author: University of Nottingham Medical School Division of Histopathy    

METHOD:

1. Wash glass slides in detergent for 30 minutes.

• Wash glass slides in running tap water for 30 minutes.
• Wash glass slides in distilled water 2x5 minutes.
• Wash glass slides in 95% alcohol 2x5 minutes.
• Air dry for 10 minutes.
• Immerse slides in a freshly prepared 2% solution for 3-aminopropyltriethoxysilane in dry acetone for 5 seconds.

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Author: University of Nottingham Medical School Division of Histopathology

Method:

1. Dissolve 5g of gelatine in 1 litre of distilled water. Do not heat over 60C.

• When the gelatine has dissolved add 0.5g of chrome alum (chromic potassium sulphate).
• Cool and dip racks of slides or coverslips into the solution.
• Drain slides and coverslips onto paper tissue.
• Dry overnight in a 37C incubator.

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Author: University of Nottingham Medical School Division of Histopathology

Method:

Many surgical specimens contain calcified areas which need to be decalcified before processing and sectioning. This is achieved as follows.

1. When the specimen is sufficiently fixed the selected tissue is placed into a labelled pot of 8% formic acid and left on the shelf in cut-up till the following day.

• Each day thereafter the tissue is assessed until it is deemed soft enough to section after processing.
• If it is not ready, the decalcifying fluid is changed and the pot is replaced onto the shelf for a further 24 hours.
• This procedure is repeated daily until decalcification is complete.

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Authors: Kai Wang & Maja Bucan

INTRODUCTION

High-density single nucleotide polymorphism (SNP) genotyping arrays recently have been used for copy number variation (CNV) detection and analysis, because the arrays can serve a dual role for SNP- and CNV-based association studies. They also can provide considerably higher precision and resolution than traditional techniques. Here we describe PennCNV, a computational protocol designed for CNV detection from high-density SNP genotyping data. This protocol extracts allele-specific signal intensities from genotyping arrays, and then integrates information on SNP spacing and SNP allele frequencies to generate CNV calls by a hidden Markov model (HMM) algorithm. Analyses of CNVs from SNP genotyping arrays will provide a more comprehensive view of genome variation, and complement current genome-wide association studies in identifying disease susceptibility loci.

OVERVIEW

CNV refers to genomic segments of at least one kb in size, for which copy number diffe

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Authors: Sohyun L. McElroy and Renee A. Reijo Pera

Corresponding Author: Sohyun L. McElroy [email protected]

INTRODUCTION

Human embryonic stem cells (hESCs) have the potential to differentiate into all three germ layers and proliferate in long-term culture in vitro. hESCs can provide a cell source for the testing of novel therapies, drug screening, and functional genomics applications. Undifferentiated hESCs can be maintained and proliferated on mouse embryonic fibroblasts (MEFs) or human feeder cells. In this protocol, we describe the culture of hESCs in feeder-free conditions on Matrigel with MEF-conditioned medium. This protocol can be used for applications such as genetic modification of hESCs without feeder cell contamination.

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