Protocol 31605: Probe Assay - Igfn1<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

Mut= 80 bp

Wt= 86 bp

Sequence

Wt Sequence (deleted region in lower case)

GATGTTTCTGGCAGGAGGTTGCCTGGCTCTGTGAGGGTGAAATAACTCCCACTCCATTTGCCTTGCATGGAACTGTAGGTCTTCTCCGAGCTGGAGTCCTACGTCCTGGCACCAGCTCCTTCGCGTACAAGCCATTACTTTCTAAGATTGTGTTGCCCATCCTCCTAGATAGGGGGAATCAAACTCATAAAATGTCATGCACTTCCTTGGTTTGGGAGAGCCCTTAGCCTCCTAACACATGCACACATGCACACATGCACACACACACACACACACACACACACACATACACACTCCTGAAGAAATCCGACTCCTACTCCATCCCTGCTTACTGCCCCCTCGACCACCACCACCACAGACCACCCAGCCACATCTTTCTCTTGGGGACACCAGGCCTctgcatgatggaagtcaggccagcaactgcttctgggtacctggaaagtatggtttccatctgacattatgaatcgcgagtaaggggatcaaagtcaggaacttcttttggaatagagagctgggatgcctacagcaggcagggtgatgtaagtttaaatccaaataacatatgttacctctctacccctatgttgcttcttccctgccacagggaaaaacgccatctttcgggcggtggtctgtggggaacccagacctgaagtgcactggcagagcagcaaaggagacctcagcaactccgccaagtaccagatatcctctgctcccggtagagaggagcacgtgcttcaggtgagccctgcagctgtttctacccatggttcacggctctcttcccagtccctaaaattcttaatggctctggtcccctggtgaagaagagatattcccacagttgggactATTCATCCTCAACCCGTCATTCAAGGTCTCCTGAGCCAGGAGAAGCCCAGATGGATTTGTTGTCATCTCCCTAATAATCAGGAGACCACCTGGACCATACTGGACCACAGTCCCAGGCCTACAGCTTTGGCTTTGGAAAGCTGGTTACTTGCCTCTTACTGTCTCAATTTCTTCACCTATGGGATAGCATAATTATAGTATACACGTTAAAGAATTATAAAGATTAAAAGTTCATGGAAAGGGTCTAGTTAGTGGTTCTAAACCTGTGAGTGTGACCCCTTTGAGGGTCAACCAACCCTCTCACAGAGGTCACATATCAGATATTCTGTACATCATATCATTACATT

This mutation is a 464 bp deletion beginning at Chromosome 1 negative strand position 135,980,217 bp and ending after 135,979,754 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37258 AGA CCA CCC AGC CAC ATC T Common A
37259 TCC AGG TAC CCA GAA GCA GT Wild type Reverse A
37260 CTG GCT CAG GAG ACC TTG A Mutant Reverse A
37261 Fluorophore-1 TCT GCA TGA TGG AAG TCA GG Quencher-1 WT Probe
37262 Fluorophore-2 TAT TCA TCC TCA ACC CGT CAT T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37258 0.40 uM
37259 0.40 uM
37260 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.