Difference between revisions of "Anorexia anx/anx mutant mice"
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| − | + | ''Anorexia'' anx/anx mice [https://www.jax.org/strain/000624 Jackson Lab strain 000624]. | |
Mutation mapped to a 0.2 cM interval residing between the markers D2Mit133 and Jojo5 chromosome 2 (Chr 2: bp 118, 889, 896–120, 175, 1081) (Lindfors et al., 2011). | Mutation mapped to a 0.2 cM interval residing between the markers D2Mit133 and Jojo5 chromosome 2 (Chr 2: bp 118, 889, 896–120, 175, 1081) (Lindfors et al., 2011). | ||
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TYRO3 protein tyrosine kinase 3 (Tyro3) mutation in anx/anx eliminatees a NlaIV restriction site. | TYRO3 protein tyrosine kinase 3 (Tyro3) mutation in anx/anx eliminatees a NlaIV restriction site. | ||
| − | * primers 5′-GATGGCGCTGAGGCGGAGCATG and 5′- | + | * primers forward 5′-GATGGCGCTGAGGCGGAGCATG and reverse 5′-CGCGGCCGGAGGTCTGGCAG ; annealing temperature 72C |
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| + | * predicted PCR product size: 280bp equal to [https://www.ncbi.nlm.nih.gov/nucleotide/1877089967?from=119630270&to=119630549&report=gbwithparts Mus musculus strain C57BL/6J chromosome 2, GRCm39], where forward primer is 119630270-119630291 and reverse primer is 119630549-119630530 | ||
* subsequent digestion with NlaIV | * subsequent digestion with NlaIV | ||
| − | * wildtype -> 175 bp fragment, anx/anx -> 250 bp product | + | * wildtype -> 175 bp fragment, anx/anx -> 250 bp product (eyeballing from Kim 2017) |
| − | [[File:anx_pcr_kim_2017.png|800px| | + | [[File:anx_pcr_kim_2017.png|800px|border]] |
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* 2024-1-18 [https://www.neb.com/en-us/products/r0126-nlaiv#Product%20Information NlaIV] ordered from NEBiolabs, along with [https://www.neb.com/en-us/products/n0556-quickload-purple-50-bp-dna-ladder#Product%20Information 50bp ladder] | * 2024-1-18 [https://www.neb.com/en-us/products/r0126-nlaiv#Product%20Information NlaIV] ordered from NEBiolabs, along with [https://www.neb.com/en-us/products/n0556-quickload-purple-50-bp-dna-ladder#Product%20Information 50bp ladder] | ||
| − | PCR: 95C for 1min, 72C for 2min, | + | ===DNA extraction from tails/spleens=== |
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| + | [https://omegabiotek.com/product-category/genomic-dna/cultured-cells-and-tissue/ E.Z.N.A. Tissue DNA Kit] yields 200 ul in elution buffer. | ||
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| + | ===PCR=== | ||
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| + | PCR master-mix: [https://www.promega.com/products/pcr/taq-polymerase/gotaq-master-mixes/?catNum=M7132 Promega Gotaq colorless master mix]. | ||
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| + | For 50 ul reaction: | ||
| + | |||
| + | PCR: 95C for 1min, then 30 cycles of: 95C for 1min, 72C for 2min. (note annealing and extension steps combined into 2 min at 72C). | ||
| + | |||
| + | |||
| + | ===PCR clean-up=== | ||
| + | |||
| + | [https://omegabiotek.com/product/pcr-clean-up-kit-e-z-n-a-cycle-pure/ E.Z.N.A. Cycle Pure Kit (V-spin)] yields 30 ul in dH<sub>2</sub>0 | ||
| + | |||
| + | ===Digestion=== | ||
| + | |||
| + | ===Gel=== | ||
| + | |||
| + | 1% agarose in TAE (1 g agarose in 100 ml TAE, microwave 2 min at 50 power; add 10 ul of SYBR safe 10000x) | ||
| + | |||
| + | load 10 ul of 50bp ladder, 10-20 ul of pre-digest and digest | ||
Revision as of 13:50, 6 June 2024
Anorexia anx/anx mice Jackson Lab strain 000624.
Mutation mapped to a 0.2 cM interval residing between the markers D2Mit133 and Jojo5 chromosome 2 (Chr 2: bp 118, 889, 896–120, 175, 1081) (Lindfors et al., 2011).
Possible genes affected
Lindsfor 2011: NADH dehydrogenase (ubiquinone) 1a-subcomplex (Ndufaf1)
Kim 2017: tyrosine kinase receptor Tyro3 which they conclude is not the anx-mutation but a strain specific modifier of anx-phenotypes
Abnormalities Table
Tyro3 Genotyping
TYRO3 protein tyrosine kinase 3 (Tyro3) mutation in anx/anx eliminatees a NlaIV restriction site.
- primers forward 5′-GATGGCGCTGAGGCGGAGCATG and reverse 5′-CGCGGCCGGAGGTCTGGCAG ; annealing temperature 72C
- predicted PCR product size: 280bp equal to Mus musculus strain C57BL/6J chromosome 2, GRCm39, where forward primer is 119630270-119630291 and reverse primer is 119630549-119630530
- subsequent digestion with NlaIV
- wildtype -> 175 bp fragment, anx/anx -> 250 bp product (eyeballing from Kim 2017)
Genotyping 1998 mice
- 2024-1-17 Tyro3 primers ordered from FSU BIO Core
- 2024-1-18 NlaIV ordered from NEBiolabs, along with 50bp ladder
DNA extraction from tails/spleens
E.Z.N.A. Tissue DNA Kit yields 200 ul in elution buffer.
PCR
PCR master-mix: Promega Gotaq colorless master mix.
For 50 ul reaction:
PCR: 95C for 1min, then 30 cycles of: 95C for 1min, 72C for 2min. (note annealing and extension steps combined into 2 min at 72C).
PCR clean-up
E.Z.N.A. Cycle Pure Kit (V-spin) yields 30 ul in dH20
Digestion
Gel
1% agarose in TAE (1 g agarose in 100 ml TAE, microwave 2 min at 50 power; add 10 ul of SYBR safe 10000x)
load 10 ul of 50bp ladder, 10-20 ul of pre-digest and digest