Protocol 42197: Probe Assay - Nelfcd<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=  127 bp

Wild Type = 142 bp

chr2:174421508+174421649 142bp GACATCTCATGGACCCACACT TGGCCAGTCTACCAGTATCG

The wt reverse primer (primer 60085) anneals over the nucleotide sequence containing mouse genomic variations rs226470986. 

Sequence

Wt Sequence (deletions in lower case):

TCTCTTGGCCATATAGAAAGGGCAGACATCTCATGGACCCACACTGTGTTTCTTTCTTTTGGGTGTTACCActgaatagacttagaggctaattctacagggtccagaatcttggctgctgttgctgtggtggtggtagagctctgcgatactggtagactggccatgctgttttgggggttcggaagggggtgctgcttcaggtaaagcttccatcttctcccaggcgttgagcctgtgcaggtgcaggagactgtggaaaaccacttgaagagtttgctgatcaagcacttcgaccctcggaaagcagattctattttcactgaagaaggagaggtgagggacgctgtccttgccccagcatgccacagccttggccagcatcctctCTGCACTCTGAGGAGTGAGGTGCTTGCATGGGAGGTTCCAGTGCTGAGGCCAGGAGCACCCAGTGTCTCCCCCGACTACTGCGTGCAGGTC

This is a 318 bp deletion beginning at Chromosome 2 position 174,421,555 bp and ending after 174,421,872 bp (mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
60084 GAC ATC TCA TGG ACC CAC ACT Common A
60085 TGG CCA GTC TAC CAG TAT CG Wild type Reverse A
60086 AGT AGT CGG GGG AGA CAC TG Mutant Reverse A
60087 Fluorophore-1 TGG GTG TTA CCA CTG CAC TCT G Quencher-1 MUT Probe
60088 Fluorophore-2 AGG GTC CAG AAT CTT GGC TG Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
60084 0.40 uM
60085 0.40 uM
60086 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.