Protocol 32408: Probe Assay - Nol9<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

>chr4:152040726+152040816 91bp CAGCCTGTCCTGTGTTCTACA ACCATGCCCTGCTCAAAAG

Mut= 95 bp

Wt= 91 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 

GCCACTGCCCTTACCCAATGAGCAGCCGTTGCGGGCTCCATGCTGTGTAGTTTGGGTTTCCTTGTCAGGTGTGGCTGGTGACATGCACTCTGAATGCAGGGATGGTTGGATTTTAATGATAGGAAGAACAGCCTGTCCTGTGTTCTACATAGTTCTGGGACAGCCAGagtcttaaggccctcaatcaagaaagaaaaatccttttgagcagggcatggtggcgcacgcctttaatcccagcacttgggaggcagaggcaggtgaatttctgagttcaaggccagcctggtctacagagtgagttccagggcagcaagggctatatagagaaaccctgtctcgaaaaaccaaaaaaaaaaaagaaaaagaaaaagaaaaaaaaggaaaagtccttttgataggtaaatcactagttagggtgtttaatggtttttttcttttcacactagggtttcactttcagtgggatctgccgtgtgacttgcgtctacggccagttggaagtatacggccatatcatcaaccaagggcagcctccccaagatgtcttttctgtctacacccactcttacctgaccatcaatggggttccgtacgcagaacccgagaaaagtgagaaggcgatcaggagggagatccgggccctgctcaagccttacacgaaactaggtaattggctcccgcttccgatgtcaaggctgatgacgaaggcttattatgggctggacgttgcgctgattcctcatggggggtgtcttacgttatgagagttgaagctggtaggtggcacacttcgatggcctgtctccacccaggtgtaccggtgaccagtgacaacctagAGCTTAGCAGTTGGGACAAGACCCTGCAGGCTCTAGAATTGTCACTGTTGCTGAGGTGTCTTGCTCTTCTTTGGCAGATGACAGAAACTGGGTGGTGCGGTACTTCCCTCCTCTGGGCTCCATTATGATTCTGGAACGGATGCAGTCTCGTTTTGTGGACTTCCTGAAGACCTATAAGTGCTCCTCATACGTGCTCCTGCAGGAGGTAAAG

This mutation is a 675 bp deletion beginning at Chromosome 4 position 152,040,765 bp and ending after 152,041,439 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39032 CAG CCT GTC CTG TGT TCT ACA Common A
39033 ACC ATG CCC TGC TCA AAA G Wild type Reverse A
39034 CCT CAG CAA CAG TGA CAA TTC Mutant Reverse A
39035 Fluorophore-1 AGG CCC TCA ATC AAG AAA GAA Quencher-1 WT Probe
39036 Fluorophore-2 ACA GCC AGA GCT TAG CAG TTG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39032 0.40 uM
39033 0.40 uM
39034 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.