Protocol 35761: Probe Assay - Cfap418<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

>chr4:10879170+10879260 91bp CATTCCCCAGTCATTCCAGT GAGTGACAGCACAAGGATGC

Mut= 86 bp

Wt= 91 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GCTTGCTCACAAAGAGTTCATCGTGTACAGAGACCATCAACAGCAGCTTCCTCCACCCCTCAGACTGTTTGTTTGTAATAAGCACTGAGTTTCCACACTGATGTTGTATCATCTGATCCCAGCCTCTGTCATTTCCTCATTCCCCAGTCATTCCAGTCAGGTCAGCCCCAAAGCCCTGCAGgttgtgacatagctccatacctgaaaagcatccttgtgctgtcactctagcagtgggacttaagcttgtggtacatctgggtttttttgtttggttggtttggtttggtttttcaagacaggatttctctgtataatcctagctgtcctggaactcactttgtagaccgtggcctcgaactcagaaatccgcctgcctctgcctcccaagtgctgggactaaaggcgtgcacgccacggctggcttggtgcatctttccttaaggtccagatgaaggttcattattgtgtgtgcatgctttcttttcagatcaacagaaacatttaaaaaagaagatgatcttgacagtcttattaatgaaatattcgaggagcctgactttgacagaaaatcttttgtaagtcactaaatgtacagtaattatttaagatgcttagactcgtcttaaagttaagtcacagtagccatctgggtatgagaattctgaggagtacaggtaaagaaaGGATTCCACTTTTTAAAATGTGGAGGGATAAAATGTTAGAAACTCTGTGTGTATTTCTGTTAACTCCTCAGTGAGAGTAAGAATTTGAAAAGCTGTTATTAGAGAAGTCAGCTCACAGACCACATTGGTCACTTAGAGTTCGTGGTAGTGTCTAAGAACGTGCTCTAAAAG

This mutation is a 505 bp deletion beginning at Chromosome 4 position 10,879,214 bp and ending after 10,879,718 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46964 CAT TCC CCA GTC ATT CCA GT Common A
46965 GAG TGA CAG CAC AAG GAT GC Wild type Reverse A
46966 TTT CTA ACA TTT TAT CCC TCC ACA Mutant Reverse A
46967 Fluorophore-1 TTG TGA CAT AGC TCC ATA CCT GAA Quencher-1 WT Probe
46968 Fluorophore-2 CCC TGC AGG GAT TCC ACT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46964 0.40 uM
46965 0.40 uM
46966 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.