Protocol 44225: Probe Assay - Gabrp<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

Mutant=  84 bp

Wild Type = 94 bp

  
>chr11:33518210-33518303 94bp TGGGTATTGCTTTTATAATTTCTCG ATTGCATCCTCTGCTCCATT

Sequence

Wt Sequence (deletions in lower case; insertions with carrots ^g^ (g insertion):

TGGGTATTGCTTTTATAATTTCTCGAGTTTTGTTTCCAACCCAC^A^taatgtagatacaccggctgcggagtctgaaatggagcagaggatgcaattggtggttagatggaggagagcttgtgtttgtgtttctatgtaagtctttctgttttttttttcttccttctcaggtgacccagttcggatagcactgactctggacatcgccagcatctctagcatctcagagagtaacatggtaaccagtgccacccccaccccttcccccgctccctctccttgctttcaattgtaatgtcctttcaagtaaatcagaaacagagtggcagatgccagagttctgtcttgtaagtggagacagtagatgtttataggttttctgggtatcaggaagggaacagactcaaaggccaggaagagagaaagtagaaaaggaagtgaccgattgactgaggaataaaaacatgtccacacgGTCTTTCAGTCGAGGGACAGCTGATAAAACTTACAATGT

 

This mutation is a 430 bp deletion beginning at Chromosome 11 position 33,517,830 bp and ending after 33,518,259 bp with a signle bp A insertion at the junction(GRCm39/mm39).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65043 TGG GTA TTG CTT TTA TAA TTT CTC G Common A
65044 ATT GCA TCC TCT GCT CCA TT Wild type Reverse A
65045 ACA TTG TAA GTT TTA TCA GCT GTC C Mutant Reverse A
65046 Fluorophore-1 ATG TAG ATA CAC CGG CTG CG Quencher-1 WT Probe
65047 Fluorophore-2 TTC CAA CCC ACA GTC TTT CAG TC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65043 0.40 uM
65044 0.40 uM
65045 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.