Protocol 32178: Probe Assay - Atp11c<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

Mutant=  113 bp

Wild Type = 117 bp

>chrX:60295009-60295125 117bp AGCAAAGAAATACCCACTGCT TCTTAAGCTGGTAATGAGCAAGG

Sequence

Wt Sequence (deleted region in lower case):

 

CAGTTTCAAAATCATTACTTAAATTGTTTGTATAAGAAAAATCTGTTTGTGTAATGGAACCATTAATGATTTCTTCAAATCTTCATGGGGATTTCAGTTAACTATATGCATGCTACAAATGCAATACTCTCAACTGTCAGTTTCAAGGAAAGTTTTAAGTTGTTTTTCTTTTCCCTttgaaatccttttgaggcagagataattatctagttgttggtagtttaaaatagtatcctatatttggtgggtaatatccatcaacatttgtaatattcgctgatccctattactgttaaacattgctgtttctggaaccagttttatatagttataaaaaaagaaataatttatatttgattatcttctgctgtcatatagacacattatgcagtacgagataccattgcactgtgtacagccgaatccattgataatctccgagcaacaattgaatgtgagcagcctcaacctgatctctacaggtaagaagaccttgcttcaccttacttatgtctgttgtctctgcttctttttcctaccagtgtagcaaagaaatacccactgcttatttaccagcttgctctttgtatgtttttataagctgttcttattccaaggAAGGGAGATGAATCAAATTAGCCTTGCTCATTACCAGCTTAAGATAAATCATTATGGACCCAAGCGTCCTATGTTTTTCTTTTCTTTTAGTTACTTGGATCAGTTTTTTAGATGGTTAACCTAAGCTTCATCTATTAGACTAATAGAGTATCTAGAGTTAATTAAAACAAAAA

442 bp deletion beginning at Chromosome X negative strand position 60,295,494 bp and ending after 60,295,053 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38525 GCA TGC TAC AAA TGC AAT ACT C Common A
38526 TCT TAA GCT GGT AAT GAG CAA GG Mutant Reverse A
38527 AGC AAA GAA ATA CCC ACT GCT Wild type Reverse A
38529 Fluorophore-1 CAA CTG TCA GTT TCA AGG AAA GTT T Quencher-1 MUT Probe
38530 Fluorophore-2 CCA GCT TGC TCT TTG TAT GTT TT Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38525 0.40 uM
38526 0.40 uM
38527 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.