Protocol 44061: Probe Assay - Adat2<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

>chr10:13559852+13559973 122bp CCCAAGGTACACAAGAATTTCA GTTTCAGGATGCCCTTAGCA

Mut= 114 bp

Wt= 122 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

TTCTGGTAAACTCAATAAGGCTTTGCTTTAGAGTGAGGTCAGTTGAAGATATATTTGAAACTAGAGCATAAGCTTATGAAATTTGAAATCTTCCATTAAAATATTCAGAAATGGTAGGTCTTTTTTTTGAGACTTATCTCCTCTGTAATAATTTAATGAAAAACAAATTTTCCCAAGGTACACAAGAATTTCACTAAAAATATAGCAAGGTTTGTGTAGTACAGTGGATTAGACATCCCTgttggtgagtttatgatatttgcactggggaactgctaagggcatcctgaaacccatgttctaatacagtcagaaatggtaggaatgccttttgaattgacattttgcatattttaaagacaccagctgtacgaaacaaacaatgtcaatacaactctctctctctcccccccccctctctctctctctcaaggctactcggcatgctgaaatggtggccattgatcaggtcctagactggtgtcatcagcatggacagagcccttccactgtattcgagcacaccgtgttgtacgtcactgtggagccgtgcatcatgtgtgctgccgccctgcgccttatgagtatcctttggcttctagacttcctgcctttaccttgcctgtgagcacaacatggccagattcacgacccattgttctttctttttagtgcttgtttttcttttcaacgttgagaatggaagccaggctcttGTACTTGCTGCATCTGCTCTGTCCCTGAGCCATAGCCGTCCTTAGCTTTCAGTTCCTTAATGTGTGGAAACAGCGTTATGAAATACACTCTACAATTTAGCCAGAAACAGGAAAACCAGTTTCTACACAGCAGAGTGACCTGTTCTTTCTCTGTATCGACAGCCATGAAAGAATTGAATACTTAAGCAAAGGGAAGAAAAGCTCTTTTGTCAGGATAAATCCAAATTAAGGTGGAGCACAGG

 

This mutation is a 476 bp deletion beginning at Chromosome 10 position 13,435,665 bp and ending after 13,436,140 bp (GRCm38/mm10). 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64690 CCC AAG GTA CAC AAG AAT TTC A Common A
64691 GTT TCA GGA TGC CCT TAG CA Wild type Reverse A
64692 CTA AGG ACG GCT ATG GCT CA Mutant Reverse A
64693 Fluorophore-1 TGG TGA GTT TAT GAT ATT TGC ACT G Quencher-1 WT Probe
64694 Fluorophore-2 AGA CAT CCC TGT ACT TGC TGC ATC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64690 0.40 uM
64691 0.40 uM
64692 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.