Protocol 36360: Probe Assay - Denn2b<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

>chr7:109557340-109557466 127bp CTTAGGTCTCAGTCAGTCTCTCCAC TGAGGAGCACCTGGGATTTAG

Mut = 145 bp

Wt = 127 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletion in lower case):

 TTACTCAGGTCTCTGAGAATGAGCGAGACAGAAAGGTTCGTGGAGGTTGGCCTGCCGTGACTAAAGAGCTATTCTTTACTTGAGGACAGCCTTAAAGATACATATTAAATGAAGGATTTCTTTTCTTTGTTTTGGACATACTAACTCAAGCTGGCCTCAGACTCACTGTGTAGCCAGTGATGACCTTGAACTCACAATCCTCTTGCCTTCATCTCCCAAGTGATGGGATTGGTATGGGAGGGGGCACTTAGAGTATTTTTGAGACTTCTGCAGGAACATAACATCAGAATGAAACACAGTCCCCCTGGTTTTTAGACCACTCACCCTTTTCCTTTTTCCTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCTCTTAGGTCTCAGTCAGTCTCTCCACCTCCAGTTTTATACCCACCAagaagtcccatctacccactcagtgacagtgaaacctcagcctgcaggtaccccagccactctaaatcccaggtgctcctcaaggaccggcattctcgaaatccttcactcctaggtcaagatccgtccccagaaacctcaccacccatttgtaccctcaaggctaccagcttcagctatttggacagaaccccttcattacgcaaaagagaggaccagaaggagactgtccagggtgcagtccaggatgtagaaggtgtggctgcttgcctcccccttgcccagagcaccccattcctaggggccgggtctcggagtgtcttgctgagttgcactggtacccgagcccatagcctgggtatccgagagaagatctcagcatgggaaggtcgccgagaggcttcacccaggatgagtctgtgtggagagaagcgggagggccctgggagcgagtggtcggtcagtgagggctgccccagtgtgggctgccccagtgtggtgccatccccctgcagctcagaaaagacctttgatttcaaaggcctccggaggatgagcaggaccttctctgagtgttcctaccccgagacagaggaggaggcagaggcacttcctggacgggactctttataccggctggagaagcgtccaggccggactgagcccagtgccttgctcagggggcatggcatcaggaaagagagctcagcagtgctgagccggatccagaagatcgaacaagccctaaaggagcagcctggccggggtctcccccagctccctagcagctgctacagtgtagaccaagggaggaggaagactggaactttgggcaccttggaggagccaacaggaactgcaagtgtaagccccagcagcagggcaggaggagtggccggagttgctggggaggcaggcccacccctggacagggaaggcagtgcttccatgaagtcagagacccctggaaatagctccagtccccagctgctgcctccgaagagctcccctgatccagctgtgaaccctgttcccaaacccaagcgtacctttgagtacgaggctgacaagaatcctaagactaagccaagcaatggtctacctccttcacccacacctgctgctccaccccccttgccttccaccccagccccaccagtcacccggagacccaagaaggacatgcgtggtcaccggaagtcacagaacaggtaaggCATGTGCTCCTGTGTCAGGAAGCAGGACCTAATGGCATTCTTTTAAATGGAAGAAGCAAGAGCAGCATGTGCTTGGGTCACAGGCGTATGCTACAAGCTCTGAAGCGCTGAGTAATTAATTGTTAGTTTGCCAGTTTGGGCAGAGAAATGGTTCTCCTGCTCCCCACTACCACACACATACCTCCTTTCATTTTTGTGCCTGAACTGGAACCTGCTATAGACCAGGCTGGCCTTAAACTTTTGCCTTGGCCTCAGGGCTGGGTTTAAAGTCATGTACCACCACCAGGCCTGTCCTCCAGCTCTTGAGTGGAAGAAAGGATGAAAGCCACTCTTGCCAGTGCTGGGAGGCTTTGTTAGTGTAGAGCCAGCTATGCTAACAATTC

This mutation is a 1,217 bp deletion beginning at Chromosome 7 position 109,556,205 bp and ending after 109,557,421 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48524 CTT AGG TCT CAG TCA GTC TCT CCA C Common A
48525 TGA GGA GCA CCT GGG ATT TAG Wild type Reverse A
48526 GAG CTT GTA GCA TAC GCC TGT Mutant Reverse A
48527 Fluorophore-1 TCA GTG ACA GTG AAA CCT CAG C Quencher-1 WT Probe
48528 Fluorophore-2 CAC CAC ATG TGC TCC TGT GT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48524 0.40 uM
48525 0.40 uM
48526 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.