Protocol 35946: Probe Assay - Fam168a<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

>chr7:100831468+100831600 133bp CAGGTAGGTACACCTGGAGGA GCAGGAATTGCTTCAGTGACA

Mut= 126 bp

Wt= 133 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CTGGGTAAGGGGCCAGAATGAGAACAGAGCAATAAGAAATGAGGGAGTAGGGGGCATCTCTACATGTACTTTCCACACAGGCAACTCTGAACCCAGGCAGGCTGTCAGCTGAAAAGGCCCCAGAGAGACTGCTTCTAGCCTCTTTTTTTGAATAGAACCCTGGAACCCAGAGAACTAACACATATCCAAATCATACTTGCAgcccatgggcaccaggactgagggagctgcctggggtcaggctctgagggtagaaccttgatctcttgtctgtcctatctgtgtttccagctggattcaacctgttcttgtttgaggaactgttactgggccctgctgtcttcaagcaatgctgttctaccaactaactagacgtctcttttattgccttggtacaggatataccgcagggacaccatacaaggtcccaccgacccagagtaatactgctccacccccctactccccatcacccaatccctatcagacggccatgtatccaatcagaagtgcctatccccagcagaatctgtatgcccaggtaggtacacctggaggacctcttctccgggagcctccagccaggatggggtggcatgggatgaggaagaaaaaacagagttaatgggcctgGCCAGCTATCACTGTAGTGTCACTGAAGCAATTCCTGCAGGGCTGCCTGGGCTGCCTTACTCCCTAAGGGTGAAATGTCAAGTCCTTTAGGTTAACAGAGCCCCAAATGTCAGAGGGCCATTAGAAATCACCCAGCATAACTCTCTTGTTGGACAAGTGGGGAAGCTGAGACTGCAGTGGAGAAAGACTTGTCTAAG

This mutation is a 432 bp deletion beginning at Chromosome 7 position 100,831,131 bp and ending after 100,831,562 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47364 CAG GTA GGT ACA CCT GGA GGA Wild type Forward A
47366 GCA GGA ATT GCT TCA GTG ACA Common A
47367 AAG GCC CCA GAG AGA CTG CT Mutant Forward A
47368 Fluorophore-1 CTT CTC CGG GAG CCT CC Quencher-1 WT Probe
47369 Fluorophore-2 CAA ATC ATA CTT GCA GCC AGC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47364 0.40 uM
47366 0.40 uM
47367 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.