Protocol 34399: Probe Assay - Niban2<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

>chr2:32905609+32905732 124bp AAAGCCCTCGTCTGGAGTG ACAGAAACCTGGGGAATCAG

Mut= 118 bp

Wt= 124 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

GGGTGTGGTTTTCTGTGACGGCCACTCACCAGCTTCCTGTCAGGAATGGGTACCTACCTCATACAGGCAGCTCATGACCAAAGCCCTCGTCTGGAGTGCGAGAGAATAAGGTGACcagccccggtttgtgtgaaaggatatttgtgctttgtgtggacggtttttcaggcgggacttgcaggcctgattccccaggtttctgtggcaacctcacactctcatgcatccttcccttgcagagaaaaccgagaagatcctcacagagttccttcgcttctatgaggaccagtatggtgtctccctcttcaatagcatgcgccatgagatcgagggcaccgggccaccgcaggcacagctgctctggcgcaaggtgagaggcatagagagggtccagcctgggctctgtaccccttctgctatgggcctttgtcttctctgggccttagtttccctatgctgctggtttggtctctaagggccttcataggttgaggataaccaccccacacttgcttcttagtctccactaccagtgccacccctaccccactgccagtgttacctaccctcactgccagtgctacccccccactgccagtgtcacccctctcccactgccagtgccacccacccccactgccagtgtcacccctctcccactgccagtgccacctctaccccactgccagtgccacccctccctcactaccagtgtcacccctccctcactgccagtgccacCCACCCCCACTGCCAGTGCAACCTCTCCCCCAATCCCAGTGCAACCCCTCCCCCAATGCCAGTGCAACCTCTTCATTGTCACAGCTACATTCTGCAGTCTTGACAGCTCAGGCCACACACCTGTCAAGAGAGAT

This mutation is a 626 bp deletion beginning at Chromosome 2 position 32,905,645 bp and ending after 32,906,270 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43508 AAA GCC CTC GTC TGG AGT G Common A
43509 ACA GAA ACC TGG GGA ATC AG Wild type Reverse A
43510 GTG ACA ATG AAG AGG TTG CAC Mutant Reverse A
43511 Fluorophore-1 CCC GGT TTG TGT GAA AGG Quencher-1 WT Probe
43512 Fluorophore-2 AGG TGA CCC ACC CCC A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
43508 0.40 uM
43509 0.40 uM
43510 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.