Protocols

Disaggregation of intact colon crypts

This protocol describes how to disaggregate whole colon tissue into a suspension of colonic crypts. This protocol was adapted from Samuel et al., Am J Physiol Cell Physiol 296: C296-C305, 2009. The sample image shows a suspension of crypts, with intact crypts outlined in red.

 

Genotyping Aag-/- Mice from Ear/Tail

A protocol for genotyping Aag-/- mice from ear punches or tail clippings. The Aag gene codes for alkyladenine DNA glycosylase, an enzyme that initiates repair of a variety of DNA lesions via the base excision repair pathway.

 

Genotyping AagTg+/- Mice from Ear/Tail

A protocol for genotyping AagTg+/- mice from ear punches or tail clippings. AagTg transgeneic animals have increased alkyladenine DNA glycosylase (Aag) activity via promoter-controlled expression of Aag cDNA.

 

Genotyping Mgmt-/- Mice from Ear/Tail

A protocol for genotyping Mgmt-/- mice from ear punches or tail clippings. The Mgmt gene codes for O6-alkylguanine DNA alkyltransferase, which converts mutagenic O6-methylguanine lesions in DNA to guanine by demethylation.

 
 

Immunofluorescent staining on paraffin-embedded tissue sections

This protocol describes the steps necessary to fluorescently detect proteins in paraffin-embedded tissue sections. After depariffinizing and rehydrating the tissues, antigens are exposed through incubation with boiling antigen retrieval buffer. Subsequently, sections are blocked with bovine serum albumin and probed with primary antibody overnight. After a series of washes, the sections are incubated with secondary antibody and detected with a fluorescent microscope.

 

Making cell line samples for proteomic analysis

This protocol describes how to make cell line lysates for downstream proteomic analysis by mass spectrometry. In this protocol, BaP is used as an example for treatment, but the process can be applied for other treatments.

 

PCR to genotype RaDR mouse from ear punch

This is the protocol to genotype for the RaDR transgene in mouse ear punches. RaDR mice were developed in the Engelward lab at MIT, and they allow detection of sequence rearrangement mutations arising from aberrant homologous recombination in situ. Please see the paper below for more information on RaDR.

 

Photolithography

Engelward Lab protocol for creating microfeature molds from silicon wafers by lithographically patterning SU-8 photoresist.

 

Preparing a Chemiresistive Sensor Based on Functionalized Carbon Nanotubes

This protocol describes how to covalently functionalize single-wall carbon nanotubes with iodonium salt and fabricate a CNT-based chemiresistive sensor for the detection of carbon monoxide.

 

Preparing Mouse Organs for Flow Cytometry

This protocol describes how to disaggregate fresh tissues into single cell suspensions for analysis by flow cytometry. The protocol was developed for analyzing the proportion of EGFP+ cells in RaDR mouse tissues, but it can be adapted to any application requiring a suspension of single cells.

 

RaDR Mouse Necropsy and Imaging

This protocol describes how to collect tissues for imaging EGFP foci in RaDR mice. Includes tips for clean excision and removing autofluorescent substances.

 

RNA-seq sample preparation for MiSeq sequencing (Illumina) using SPIA amplification method

RNA extraction from tissue or low yield samples and preparation of RNA samples for RNA-sequencing (RNA-seq) using NuGEN’s SPIA (Single Primer Isothermal Amplification) amplification technology.

 

Single Cell Microarray for High Throughput Detection of DNA Damage

Motivation
• DNA damage is associated with an increased risk of cancer, aging and disease
• Few methods are available for high throughput analysis of DNA damage
• This protocol is based upon the traditional comet assay wherein DNA migrates more
readily through a matrix when damaged
• 96 samples can be processed in parallel
• Throughput and sensitivity are increased compared to the traditional assay

 

Staining for Ki67 in paraffin-embedded tissue sections

This protocol describes how to immunofluorescence stain for Ki67, a marker of cellular proliferation, in paraffin-embedded tissue sections.

 

Treating TK6 Cells with Lanthanide Chlorides (for use with CometChip protocol)

Description of how to prepare the cells and lanthanide solutions used for studying DNA damaging potential of these metals.

 

Western Blot with BioRad Protean 3 Cell

This protocol describes how to perform a Western blot using the BioRad Protean 3 Cell system, one of many available Western blot assemblies. The basic steps of this protocol can be adapted to any Western blot assembly, but we recommend reading the instructions for your system first.

 
 
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