Protocol 33826: Probe Assay - Atp7a<em1(IMPC)J>
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

>chrX:106085385+106085536 152bp CATCTGTGGTGAGTGCCAGT GGTCATCCCTTCCACTTTCA

Mut= 165 bp

Wt= 152 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

GGCCCCGTAAGATTTCTTTCATCCACACTGGTATGTCAACTGGTATTGTCGTTATGCAGGTCTTGTTTAGGTTCCATGGATGCAGCTATAAAAATTTTACCCACTCATATTCAATGTTCCCTGAGCTTTAGGTATAGGATTTATGTTACAGATGCATCAGTTGTGGATGAGGACCTCATGgtactcattttgaccagttgttgatttctgtaattgtccccatctgctgcagaaaagaaaacgtctttgatgagaagtgagagctacacctagcagggaaggccagcatgtgaactaagtgtctggagctaggcattggaatgtgaaatggctcaggaggacttggttcttgataattgatagtcttaattcctatgtcatacagctgagttaaacttctgaaatttctttcccaaaggtttcactagaagaaaagagtgcaaccattatttatgaccctaaacttcagactccaaagaccctccaagaagctatcgatgacatgggctttgatgctcttctccacaatgctaaccccctccctgtcttaaccaataccgtgttcctgactgttactgctccactgactctgccttgggaccatatccaaagtacattgctcaagaccaagggtgtgactggtgttaagatttcccctcagcaaagaagtgcagtagtcaccataattccatctgtggtgagtgccagtcagattgtggagctggtcccagacctcagtttagacatgggaactcaggagaaaaagtcaggagcttgtgaagagcacagcacgcctcaggcaggggaagtcatgctgaagatgaaagtggaagggatgacctgccattcatgtactagcaccattgaaggaaaagttggaaagctgcaaggtgttcagcgcattaaaggtaattttttttttgctcattttataaagctattctttgttttgatctttaaactttctgctttcttcccaagtatgagtttcagatccgtgggcctagaattgcttcctgcttcattgaagttagaagtgtagcatagtcccaggattaatgtcagtaaagttaatgattataatgaaaggttatgagagttgaacttcagtttagcaggaaaggcactgtgGTAGTGATTTGTGGAGTTTAGGCTTCCTGAACAGGCTGCCTTAGGCATATTTTATCTCTAATATATTTGTACTGGAGTGATGCAGAAACATGGAGACCATAGGAGCAGTGGTCATGTTGCTTGTTTCCAAAGTATGTCAATC

This mutation is a 951 bp deletion beginning at Chromosome X position 106,084,876 bp and ending after 106,085,826 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42276 CAT CTG TGG TGA GTG CCA GT Wild type Forward A
42277 GGT CAT CCC TTC CAC TTT CA Wild type Reverse A
42278 TTG TCG TTA TGC AGG TCT TG Mutant Forward A
42279 TCA GGA AGC CTA AAC TCC ACA Mutant Reverse A
42280 Fluorophore-1 AGA TTG TGG AGC TGG TCC C Quencher-1 WT Probe
42281 Fluorophore-2 TGA GGA CCT CAT GGT AGT GAT TT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42276 0.40 uM
42277 0.40 uM
42278 0.40 uM
42279 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 40.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.