Protocol 32241: Probe Assay - Dnajb5<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

>chr4:42970045+42970140 96bp TGCTTGTGTGTCTGTCTTTCTGT GGGAGGAAGATGATGTACACG

Mut= 95 bp

Wt= 96 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

CAGACTAGCCTAAAACTCTCAATCCTCCTACTCAGACTTTATAGAGTACTGAGATTACAGACATGCAACACCATGCCATGTACACATTCCTAAATAGATATCCAATTGAGTAGAAATTAACCTCGTCCAAAGCCTTTCTTATTCTGTCAGCGTGGTCCTCTCTGGGGGTTAGAGACTGCTTGCTTCCCTCAGCCCAGGAGCCAGCATTTAGTGGCAGAGGgaagggactggggtggggcgaggatggaccacactgcccctaacttctcgtcccctttaggcctcaagaccggcggtggttcctcaggtggttccggtggctcctttcactacacctttcatggggaccctcatgccacctttgcttccttctttggtggctccaaccccttcgatatcttctttgccagcagccgctccactcggcccttcagtggctttgaccctgatgacatggatgtggatgaagatgaggacccatttggtgcctttggccgctttggcttcaatgggctgagtaggggtccaaggcgagccccagagccactgtacccccggcgcaaggtacaggacccccctgtggtgcacgagctgcgggtgtccctggaggagatctaccatggctccaccaagcggatgaagatcacaaggcggcgccttaaccctgacgggcgaactgtgcgcaccgaggacaaaatcctgcacattgtcatcaaacgaggctggaaagaaggtaccaagattaccttccccaaagagggcgacgccacacctgacaacatcccagccgacattgtcttcgtgctcaaagacaagcctcatgcacacttccgccgagatggcaccaacgtgctctacagtgccctcatcagcctcaaggaggtgtgtgtgtgtgtgtgtgtgtgtgttcgcacgtgtgtatgtatgtgcttgtgtgtctgtctttctgtctgttagagggtgataggaatcatttcctgcctgcctgccatggtCTTTTCCCGTGTACATCATCTTCCTCCCTCTCCACTCTTTGCCTACTTTTCCCTCACTCTGTGTTATTGTTTTCCAGGCGCTGTGTGGCTGCACCGTGAACATTCCCACCATTGATGGCCGAGTGATCCCTTTACCCTGCAATGATGTTATCAAGCCAGGCACCGTGAAAAGACTCCGTGGGGAGGGACTTCCCTTCCCCAAGGTGCCCACTCAGCGGGGAGACCTCATT

 

This mutation is a 775 bp deletion beginning at Chromosome 4 positive strand position 42,956,466 bp and ending after 42,957,240 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38657 TGC TTG TGT GTC TGT CTT TCT GT Wild type Forward A
38658 GGG AGG AAG ATG ATG TAC ACG Common A
38659 GGT CCT CTC TGG GGG TTA GA Mutant Forward A
38660 Fluorophore-1 CTG TTA GAG GGT GAT AGG AAT CAT TT Quencher-1 WT Probe
38661 Fluorophore-2 TTT AGT GGC AGA GGC TTT TCC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38657 0.40 uM
38658 0.40 uM
38659 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.