Protocol 35995: Probe Assay - Retreg1<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

>chr15:25895129+25895234 106bp AAGCCTCACTGAAAGGTAGGG ATTCAGCGATATGCCTCTGC

Mut= 109 bp

Wt= 106 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (GTATA insertion):

 TGAAGACTGCACCATGCCAACCTCCCCTCCCATACCATTGCCATTACCTCGACTTGAGCAGTCATGTTTCTGTCCTTTCTGAAGTGTGATGGTTTCCACCTCTTGTCAAATTTCATACTGACTCCTTGACAGCACTTTTTTCTTAAGTGGACATGTTTTCTTCCCACTGGGCAGGACTGTGTTTCGGAGTGTCCATCCTGCCTTTGTGATACGTAC^tcaagggttttatagactatcacctgcatccttcgcaatgatgtacgcagatactgtatgggttgacagtgtctagcacctgagatgtgagcattccgcttgcacaagtgtgggaggttgctaagctggcaaatgctcctggggagtgacagcttgctcacctttcacattttgctttccgttgcaggtgcccagctgtggagaagcctcactgaaaggtagggagtttatccttcaacaatgtggcttatggatggcagggcagtgg^CGTGGGGAGGTGATTTGAGTTGCAGAGGCATATCGCTGAATTCATGTTTTAGCGACAGTGACCTAAGTCCCTATAATGACTTGCAAGAGTGTGCAAAGTGAAGCTGAAGCTGAGGACAAGACGGGGAAGTCACTCGAGGGATAAATGCATTTCTCAGAGTGGCTGGGCAGCGGATTTGTGCGGGAGGATGCAGCAGGGAGGGAGAGGGGCCTTTCTGTTTGATGTCTTCTCTCAGATTC

This mutation is a 268 bp deletion beginning at Chromosome 15 position 25,894,926 bp and ending after 25,895,193 bp (GRCm38/mm10). In addition, there is a 5 bp (GTATA) insertion at the deletion site.  

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47522 AAG CCT CAC TGA AAG GTA GGG Wild type Forward A
47523 ATT CAG CGA TAT GCC TCT GC Common A
47524 GGA CAT GTT TTC TTC CCA CTG Mutant Forward A
47525 Fluorophore-1 CTT CAA CAA TGT GGC TTA TGG A Quencher-1 WT Probe
47527 Fluorophore-2 CTT TGT GAT ACG TAC GTA TAC GTG G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47522 0.40 uM
47523 0.40 uM
47524 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.