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added more genotyping details
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Line 1:
−Anorexic anx/anx mice [https://www.jax.org/strain/000624 Jackson Lab strain 000624].+
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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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Line 17:
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′-CGCGGC- CGGAGGTCTGGCAG ; annealing temperature 72C
+* primers forward 5′-GATGGCGCTGAGGCGGAGCATG and reverse 5′-CGCGGCCGGAGGTCTGGCAG ; annealing temperature 72C
+* 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|thumb]]
+[[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, 30 cycles
+===DNA extraction from tails/spleens===
+[https://omegabiotek.com/product-category/genomic-dna/cultured-cells-and-tissue/ E.Z.N.A. Tissue DNA Kit] yields 200 ul in elution buffer.
+===PCR===
+PCR master-mix: [https://www.promega.com/products/pcr/taq-polymerase/gotaq-master-mixes/?catNum=M7132 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===
+[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