Protocol 35783: Probe Assay - Dnaaf1<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

>chr8:119578941+119579060 120bp GACCAATGGAGAAAGGAGGA CTGGGATTGCTGCTGTGA

Mut= 103 bp

Wt= 120 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CCAGTCTACCCGAATCAGTAGTCTCCAAGTTCAATGAGAGAGAGAGTTCATAGTTCAAAAGCTATGGCAGATATGATAGAGAAGACAGCTGGTGTCACACCTCTGGCCTCTGTGTGACCTTGCATACCCAGGAACACACACAAACAGACCAATGGAGAAAGGAGGACGAGGAGGACccgatgtgaggcttcagattgaaaactttagacagaatggatggagaaaagagcctgagcgggtgccatggctcacagcagcaatcccagcggtggcgcaggcaggagagccacaagcttgaggccagcctgggttacctactgagttcccagctgaccctgtctccaaactaaatggagccaaatacaaacccttaactattttttttcacagacgacaagctgaagtgagcgtcttgtgttttgcttttcaacaggatgacgaaacagttccttcaaaagctctgcaagcagcacaagctgtacgtcacccccgccctgaacgacaccctgtatttgcactttaagggtgagtcccaggcgagtctatagccagcgtcctgcagaaaggggggagctggagagcatggcttgcagaggttgtcttcgcgctcagacgctatgcaagtggagctcggtcttgactgagttcctaaagggcCTTCTGAGTACAGTGGAGCTGGCCCGCCTTCTTTGGTTGCTTGGTTTTTTTCTTCCTTGCAAAATAGGGATGGAACCCAGAACCTCAAACATGCTAAGCAGGCACACTGCCACCAGATACAACTTCAGTTAGCCCGCCTTCCCCATTTTATTTTGAAACAAGGCTCACCAAATTGCCCTGGC

This mutation is a 481 bp deletion beginning at Chromosome 8 position 119,578,971 bp and ending after 119,579,451 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47018 GAC CAA TGG AGA AAG GAG GA Common A
47019 CTG GGA TTG CTG CTG TGA Wild type Reverse A
47020 CCA TCC CTA TTT TGC AAG GA Mutant Reverse A
47021 Fluorophore-1 ATG GAT GGA GAA AAG AGC CTG Quencher-1 WT Probe
47022 Fluorophore-2 AGT ACA GTG GAG CTG GCC C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47018 0.40 uM
47019 0.40 uM
47020 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.