Ideally, mice and rats should be less than 21 days old for genotyping. Extraction protocol notes the protocol is flexible and can be optimized. Ez fast tissuetail pcr genotyping kit ez bioresearch. Dna extraction from mouse eartail to genotyping karine. Brandon willis summary reagents and materials protocol reagent preparation reagent 1 summary. I recommend mouse genotype for any mouse genotyping needs.
Genotyping is the process of detecting the presence, or absence, of specific dna sequences in a particular organisms genome. I am genotyping knockout mice with a pcr protocol that has worked very well up until this month. Label the mice s number, sex and ear punch label l, r, ll, rr or lr on the micro centrifuge tubes. Avoid repeated freezing and thawing of nucleotides. Animal research advisory committee guidelines oacu. As with any procedure, the specific method of tissue collection must be detailed in the approved iacuc protocol.
Although you may be able to detect up to 5 kb sequence from dna samples prepared by kapa mouse genotyping kit if a highly optimized condition is employed, saltingout method is far more suitable for such applications. Genotyping by southern blot there are two straightforward ways of genotyping baygenomics mice by southern blot. Mouse tailing pups are tailed for dna and toed for identification between 814 days of age. Analysis of 890bp genomic dna derived from 2mm mouse tail snips. Omniprep genotyping dna extraction kit from mouse tail. Add 600 microliters of tnes and 35 microliters proteinase k 10 mgml. The mouse tail biopsy procedure is the most typically used procedure to obtain tissue for a southern blot test. Collection of tissues for genotyping asc research support. Protocol for genomic dna isolation from mouse tails to compare the dna quality resulting from the ethanol method with the standard phenolchloroform method 1, we extracted dna from the same set of samples using the two methods. Genotyping baygenomics mice university of california, davis.
Dna extraction from mouse tailtips for genotyping youtube. Tail biopsies should preferably be taken from animals between the ages of 8 and 12 days of age because there is a lack of nervous. In a young mouse less than or equal to 3 weeks of age, the tissue near the tip of the tail is soft and the bones have not completely mineralized. Dna is from a crude lysis method involving a 12 mm tail sample, protocol below. Dna isolation from mouse tail samples add 100ul lysis solution per 12 mm. Iacuc policy for identification and genotyping methods in mice.
Basic protocol 1 tissue sampling methods and procedure there are several ways to obtain dna for mouse genotyping. Outsourcing is costly and may not be as fast as desired, especially when setting up time sensitive experiments. Dna was extracted from eight mouse tails and pcr amplification of the three target genes was carried out using the ez fast tissuetail pcr genotyping kit. When collecting tail tips, clean tools with 70% etoh between animals. Please acknowledge the genotyping core laboratory in all publications and grant applications if you are using equipment, technologies, expertise, or data generated at the lab. The patentpending components developed by scientists at viagen biotech inc. Our advanced buffer and antibodymediated hot start technology allows robust performance and high sensitivity across a wide range templates, including gc and atrich sequences. Mouse metabolic phenotyping centers mmpc protocols 031215 1 of 3 pages standard pcr genotyping protocol version. Although tail biopsy is a safe, effective and humane procedure for mice and. Purpose researchers must consider all sources of dna to perform genotype analysis, including alternatives to invasive procedures such as tail biopsy. Standard pcr conditions for genotyping of mutant mouse dna extracted from somatic e. The tail snip is not useful for identification of mice.
Pick the mouse up by the tail and firmly grasp majority of the loose skin on the. A summary of the most pertinent points when describing genetic variation is included chapter 17. Genotypingby sequencing for plant analysis is also included chapter 16. Amplification of the sexdetermining target 144 bp and 166 bp doublet for males, and no product for females and internal control 527 bp fragment was performed using platinum ii taq hotstart dna polymerase. Hold mouse firmly at base of tail with one hand, with the other cut off 0. Slow and laborious genotyping procedures add to the hassle. Prepare paper towels and block the air grilles to prevent samples 9. Universal mouse genotyping protocol mouse genetics core.
Dna in crude extracted from tail, ear, or toe tissue, with specific authorizations according to your local ethical regulations. Genotyping protocols in general university of calgary in. Euthanasia of rodents using carbon dioxide, 102319 revised. Authorship should be considered when lab members are significantly involved in developing the experimental design or contribute to the publication content by advancing it. The tail of a mouse is composed of bone, cartilage, blood vessels, and nerves. Proteinase k is stable in directpcr reagents for 24 hrs. 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. Mouse genotyping perelman school of medicine at the. Uc davis 2795 2nd street, suite 400, davis, ca 95618. Since genes can influence a mouses phenotype, being able to probe an individual mouses genetic makeup, or genotype, is critical for attributing a. Dna extraction from mouse tail to genotyping no organic solvents extration 1.
Nih arac guidelines for the genotyping of rodents recommends that rodent tail biopsies be performed at 10 21 days of age before the vertebra are calcified. Guidelines for genotyping laboratory mice and rats. All is needed is the complete sequence of the trapped intron, and the sequence of the vector used to trap your geneofinterest. This method refers to the amputation of the distal 5mm or less of tail tissue in order to obtain dna for genotyping. The tubes must have tightfitting caps, so that there are no leaks in steps 3 and 7 below. Tails can be stored at frozen in pbs or pbnd until use. Select the according protocol on the thermal cycler then put it back to the fridge after the cycle is done. The mbp offers a variety of services for mouse genotyping, customized to meet the needs of our clients.
Genomic dna extracted by kapa mouse genotyping kit works best when pcr products are in the range of 150 bp 1 kb. One approach is to choose a restriction endonuclease that will cut in the vector but not in the. You provide us with the sequence of your primers, or we design an assay for you, and we validate the pcr assay at no cost to you. Ultrafast genotyping protocols often require realtime pcr instruments and commercial reagents that may not be economical or practical. Obtaining tissue from a mouse or rat for dna analysis via tail biopsy is a safe, effective, and humane. Ge notyping from mouse tail using platinum ii hotstart. Pick the mouse up by the tail and firmly grasp majority of the loose skin on the back of the mouse using thumb and index fingers while still holding the tail near the. This protocol yields a highly purified dna preparation from mouse tail biopsies. The method selected depends upon several parameters, including the established practice in your laboratory or institute and the. Last, but certainly not least, it is critical for genotyping findings to be reported in a clear and unambiguous fashion. Pcr biosystems offers a range of polymerases that are designed to perform in mouse genotyping studies with high efficiency and improved tolerance to common inhibitors that may exist following dna extraction steps.
Genotyping by sequencing for plant analysis is also included chapter 16. The accustart ii mouse genotyping kit is designed for fast and easy preparation of pcrready dna extracts and endpoint pcr analysis from tissues such as tail snips and ear punches commonly used for genotyping knockout and transgenic animals. When repeated tail clips are necessary, regardless of age, they require general anesthesia as described in the. Do not perform pcr in a ventilated hood as it increases the risk of crosscontamination. This protocol was developed from the nih guidelines for the genotyping of mice and rats revised 51210. The four protocols presented in this paper are optimized for the most common samples used for genotyping transgenic mice. The first protocol is for genotyping a subset of marker positions genomewide using restriction digestion, and the second is for preparing inexpensive pairedend wholegenome libraries.
Gentra puregene mouse tail kit for isolate genomic dna. Dna in crude extracted from tail, ear, or toe tissue, with speci. The procedures for tail biopsy for dna analysis or genotyping must be described in an approved iacuc protocol. Dna extraction from mouse eartail to genotyping no organic solvents extration obtain the last 2 mm of the ear or tail tissue and place directly into 75. Genotyping rodent tail snipping for genotyping policy. We provide a complete mouse genotyping service, from dna extraction to a comprehensive genotyping report. Obtain the last 2 mm of the tail and place directly into 75 l alkaline lyse reagent in a pcr tube. Lane 1 azura mouse genotyping kit 1 hour duration lanes 2 and 3 conventional proteinase k and standard taq polymerase workflow 6 hour duration lanes 4, 5, 6 and 7 mouse tail snips of 1mg, 2mg, 3mg and 4mg respectively. At this age, the tail tissue is soft vertebra are not yet calcified and the yield of dna is high. Tissue collection for genotyping mice and rats, 022818. Dna extraction from mouse tail to genotyping no organic. Genotyping methods and protocols pdf arslan library. Basic genotyping protocol university of washington. There are several ways to obtain dna for mouse genotyping.
You have competitive prices, work amazingly fast we often wonder how you are able to do the genotyping so quickly, and even more importantly you provide excellent customer service. The three amplified dna fragments 220 bp in lanes 18, 498 bp in lanes 18 and 670 bp in 18 were analyzed on a 2% agarose gel. Tissue collection for genotyping mice and rats, whether performed by dact personnel or investigators, will follow the protocol described here unless alternate methods are described in an iacuc. Protocol for genotyping knockout mice stephanie kim and guowei sean zuo 102009 i. Genotyping rodent tail snipping for genotyping policy printerfriendly version an exception to a policy must be described and justified in the animal protocol and approved by the full iacuc at a convened monthly meeting.
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