Apc min genotyping protocol pdf

Taqman snp genotyping assays thermo fisher scientific. Both reduced and increased vitamin content resulted in a 70 or 80% increase in polyp number in the proximal third sb1 of the small intestine p min mouse model of intestinal cancer, genetic ablation of stat3 reduced the multiplicity of early adenomas. Trisomy represses apc min mediated tumours in mouse models. Genotyping by sequencing sequencebased genotyping methods. Heterozygotes of this strain develop anemia and are highly susceptible to spontaneous intestinal adenoma formation. Apcmin mice have provided an example of a locus modifier of min1. Mouse tailing pups are tailed for dna and toed for identification between 814 days of age. Markedly enhanced colon tumorigenesis in apc min mice. For example, progeny of apc fle114 min x apc fle114 min that didnt carry min min genotyping protocol yields wt were apc fle114fle114 homozygous. Various c57bl6japcminj mouse breeding and genotyping. Does anyone have knowledge on genotyping for apcmin mice. Basic genotyping protocol university of washington. Clusterin as a biomarker in murine and human intestinal.

However, loss of stat3 promoted tumor progression at later stages, leading to formation of invasive carcinomas, which significantly shortened the lifespan of stat3. Taqman snp genotyping assays are the worlds largest collection of singletube, readytouse snp assays available. Does anyone have a good protocol for genotyping apcmin mice. Dna extraction from mouse tail to genotyping no organic. Markedly enhanced colon tumorigenesis in apc min mice lacking. It has applications in crop improvement programs by enhancing identification of superior genotypes. A practical approach to microsatellite genotyping with. In this study, a quantitative realtime pcr analysis of the. Digestion of mouse tails for dna genotyping preparation of genomic dna from mouse tails. Als, adjust mgcl 2 to increase reaction or decrease non specific amplifications. Genotyping protocols database c57bl6japcminj stock no.

Stat3 is a negative regulator of intestinal tumor progression. Genotyping by sequencing, or nextgeneration genotyping, is a genetic screening method for discovering novel plant and animal snps and performing genotyping studies. Both suboptimal and elevated vitamin intake increase. We have developed a simple method for multisample or multilocus snp genotyping using allelespecific primers asp. Dna extraction from mouse tail to genotyping no organic solvents extration 1. All assays are developed using life technologies robust bioinformatics assay. The genotyping protocol s presented here have been optimized for reagents and conditions used by the jackson laboratory jax. For each dna sample, realtime pcr was set up containing three primers a specific primer for wildtype and apcmin. For some applications, such as genotype screening and genetic mapping, sequencebased genotyping provides a lowercost alternative to arrays for studying genetic variation. Briefly, an oligonucleotide primer apcmutant was designed so that the min mutation which converts codon 850 from a.

When the shapirowilk test indicated nonnormally distributed data, a manual. Some platforms permit users to pick custom snps but the highest throughputs are available only in fixed contents. Tails can be stored at frozen in pbs or pbnd until use. Here we report a reproducible method for genotyping mice with dna extracted from fecal samples. For example, progeny of apcfle114 min x apcfle114 min that didnt carry min min genotyping protocol yields wt were apcfle114fle114 homozygous. Does anyone have a good protocol for genotyping apc flox. Suppression of intestinal polyposis in mdr1deficient. This technique employs a combination of pcr amplification and restriction enzyme digests. This video will discuss some of the principles behind snp genotyping, give an introduction to several common snp identification methods, and finally, some applications of these techniques. As well as gene specific elements, the targeting cassette contains many common elements e. Plastic beakers labeled by number one beaker per fish to be genotyped b. Gstpnull apc min mice also had significantly decreased survival, based on morbidity relative to gstpwt apc min median 21. More specifically, we 1 improved the design of allelespecific primers, 2 established a method to detect pcr products optically without electrophoresis, and 3 standardized pcr conditions for parallel genomic assay using various allelespecific primers. The protocol for detection of the min genotype has been described previously 32.

The mice were genotyped by a pcr assay 27, and both genotypes were used in the experiment. Taqman snp genotyping assays university of florida. Thermocycler tubes typically 8 or 12tube strips numbered so that each plastic beaker has a corresponding tube c. Thermo scientific phire animal tissue direct pcr kit. Genotyping of apcmin mice was performed using the standard pcr protocol provided by jackson laboratory. To genotype animals, jax recommends researchers validate the assay independently upon receipt of animals into their facility. Obtain the last 2 mm of the tail and place directly into 75 l alkaline lyse reagent in a pcr tube. With high numbers of breeding programs, it is important to have a simple method that can be used to genotype the mice noninvasively. Protocol for genotyping knockout mice stephanie kim and guowei sean zuo 102009 i. Universal mouse genotyping protocol mouse genetics core. Multiple formats will be required to carry out most projects targeting.

Protocol for genotyping knockout mice university of chicago. End point analysis assay apc min alternate1 version 5. A mouse model for intestinal and mammary tumorigenesis. For restriction digests and southern analysis, there should be enough dna for 12. Sorry that this sounds like a complete pain, and we seriously regret that we never got a genotyping protocol that worked. Each assay allows genotyping of individuals for a single nucleotide polymorphism snp. Many different genotyping approaches are available low to high throughput 2. Stat3 is a negative regulator of intestinal tumor progression in apcmin. Brandon willis summary reagents and materials protocol reagent preparation reagent 1. The genotyping protocols presented here have been optimized for reagents and conditions used by the jackson laboratory jax. In the apc min mouse model of intestinal cancer, genetic ablation of stat3 reduced the multiplicity of early adenomas. Clusterin as a biomarker in murine and human intestinal neoplasia xiaodi chen, richard b. I did find that all apc min mice had polyps in small intestines and found. Paracrine cyclooxygenase2 activity by macrophages drives.

For those familiar with pcr genotyping, this method differs from the typical protocol by utilizing a unique enzyme klentaq, 30mer primers, and a 68 annealing. Trisomy represses apc min mediated tumours in mouse. Gstpnull apc min mice had an altered phenotype was seen in the greatly increased frequency of rectal prolapse and bleeding 63% vs. Incubate at 55c for 612 hours in heat block or water bath. The taqman snp genotyping assays library consists of two collections of human assays and one of mouse assays, and can be supplemented with assays designed using our custom taqman snp genotyping assays service. Snp genotyping involves determining the specific sets of variants, in this case snps, present in an individual. Primer combination expected bands genotype for southern blot analysis please. Synergism between krasval12 and mutant apc accelerates murine.

Both reduced and increased vitamin content resulted in a 70 or 80% increase in polyp number in the proximal third sb1 of the small intestine p jun 11, 20 genotyping by sequencing is a robust,fast and cheap approach for high throughput marker discovery. Sequencespecific target amplification by polymerase chain reaction pcr is the method of choice for analyzing small numbers of snps, short or long vntrs, and short or long indels. We have several questions that we are trying to get answered and would greatly appreciate answers. B e tai ndmso isan d ardiz du to hig gc con en pomo r r gio oc l m yb w hout.

Suppression of intestinal tumorigenesis in apc mutant mice by. Ssr genotyping involves the design of dnabased primers to amplify ssr sequences from extracted genomic dna, followed by amplification of the ssr repeat region using polymerase chain reaction, and subsequent visualization of the resulting dna products, usually using gel electrophoresis. Make a cocktail of atl with 180ul buffer atl and 20ul proteinase k. Genotyping protocol provided by donating investigator genotyping by pcr protocol. U s etouch dow n cyclin gp rotoco lf i t1 0 ycl anne 65 oc dec sin mpe ure b. My laboratory is starting a c57bl6japcminj mouse the jackson laboratory breeding program. This protocol is designed to detect sequences in the murine genome by polymerase chain reaction amplification, and is adapted from stratman and simon transgenic res. Noninvasive techniques these methods of dna sampling produce minimal animal discomfort.