Difference between revisions of "Anorexia anx/anx mutant mice"
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* 2024-1-17 Tyro3 primers ordered from FSU BIO Core | * 2024-1-17 Tyro3 primers ordered from FSU BIO Core | ||
| − | * 2024-1-18 [https://www.neb.com/en-us/products/r0126-nlaiv#Product | + | * 2024-1-18 [https://www.neb.com/en-us/products/r0126-nlaiv#Product-Information NlaIV] ordered from NEBiolabs, along with [https://www.neb.com/en-us/products/n0556-quickload-purple-50-bp-dna-ladder#Product-Information 50bp ladder] and purple dye. |
* 2024-6-6 [https://www.promega.com/products/pcr/taq-polymerase/gotaq-master-mixes/?catNum=M7132 Promega GoTaq colorless master mix] ordered from Fisher (suspect green master mix interferes with digest) | * 2024-6-6 [https://www.promega.com/products/pcr/taq-polymerase/gotaq-master-mixes/?catNum=M7132 Promega GoTaq colorless master mix] ordered from Fisher (suspect green master mix interferes with digest) | ||
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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. | [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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| + | Measure DNA quantity/quality with Nanodrop. | ||
===PCR=== | ===PCR=== | ||
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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: 95C for 1min, then 30 cycles of: 95C for 1min, 72C for 2min. (note annealing and extension steps combined into 2 min at 72C). | ||
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| + | ===Gel=== | ||
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| + | 2% agarose in TAE (2 g agarose in 100 ml TAE, microwave 2 min at 50% power; add 10 ul of SYBR safe 10000x) | ||
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| + | load 10 ul of 50bp ladder; 10 ul of PCR product + 2 ul purple loading dye | ||
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| + | run at 90V for 30 min | ||
===PCR clean-up=== | ===PCR clean-up=== | ||
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===Digestion=== | ===Digestion=== | ||
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Latest revision as of 09:58, 24 October 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
Lindfors 2011 PMID 22025706: NADH dehydrogenase (ubiquinone) 1a-subcomplex (Ndufaf1)
Kim 2017 PMID 28093506: 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 (C19T-Tyro3 mutation) in anx/anx eliminates 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 and purple dye.
- 2024-6-6 Promega GoTaq colorless master mix ordered from Fisher (suspect green master mix interferes with digest)
DNA extraction from tails/spleens
E.Z.N.A. Tissue DNA Kit yields 200 ul in elution buffer.
Measure DNA quantity/quality with Nanodrop.
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).
Gel
2% agarose in TAE (2 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 ul of PCR product + 2 ul purple loading dye
run at 90V for 30 min
PCR clean-up
E.Z.N.A. Cycle Pure Kit (V-spin) yields 30 ul in dH20