Protocol 43772: Probe Assay - Fam163b<em1(IMPC)> Alt1
Version 2.0

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

  
>chr2:27113699-27113795 97bp AGGCGGATGCCTAGTCTGT GCCTCTATGCAAAGCCACTA

Mutant=  96 bp

Wild Type = 97 bp

Common Forward Primer (62628) anneals over the sequence containing genomic variation rs257721679.

Sequence

Large deletion:  Mutant sequence with junction in uppercase

aggcggatgcctagtctgtggtgaacacattctgggggattgccttctCGgcacagactctccctggaagcagatggagtccaccttcccagatca

 

This mutation is a 3410 bp deletion beginning at Chromosome 2 position 27,110,337 bp and ending after 27,113,746 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
62628 AGG CGG ATG CCT AGT CTG T Common A
62629 GCC TCT ATG CAA AGC CAC TA Wild type Reverse A
62631 Fluorophore-1 CTT CTC CCT CTT GAT GGT CTT GTA GA Quencher-1 WT Probe
62632 Fluorophore-2 ATT GCC TTC TCG GCA CAG ACT C Quencher-2 MUT Probe
63891 TGA TCT GGG AAG GTG GAC TC Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
62628 0.40 uM
62629 0.40 uM
63891 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.