Protocol 34787: Probe Assay - Ier5l<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=  117 bp

Wild Type = 116 bp

>chr2:30472246-30472361 116bp GACGTTCCGCTCTCCTGAGT GAGGAAATGTGACCGAAACG

Sequence

CGCGCACCTGCCGCAGGTGAGGCCCAACCATGCGGAAGGGGGCGGAGCCATCTCCGCTGCGCTCAGGTGAGCCGACCCCCAGCTCGCAGGAGCCACGTCCAGGAAAGCTACGAATTTCGACGTGGCTCCCAAGAGGCGACCCAAAGCTGAGAGAGACTACCTCGGAGGCTCCTAGTCCcacgaggtgccgcggccccaggagaacattcgaggcaacggcggcgttggaggcccggggtccgttcgttatttgaatagcacaaaggaaccgccttggtctgattggccagtcaagtggtcccggggagggcagtggctgtcagtcggaaggctctacacgcttctattggctaatctacagtggggcggggcgctgctgcccagctaaatagttggggccggggccggcggaggctcattgctttggcgccgtctggggagcgtgagcctgcgggtggcgcgcaacgcatggtggcgacttctctcggagtgtcgccgacccgccgacccgggctcggttccctgtgcctggctcgcccgggcgaccaactggagacacacgcaaggagcagcattcgcagcaccagaggcggctgctccgccgcccgcgtctccgcgggagcatggagtgcgccctggacgcccagagcctgatcagcatctccctgcgcaagatccacagctcccggacccagcgcggcggcatcaagctgcacaagaacctcctggtgtcctacgtgctccgcaacgcgcgccagctctacctgagcgagcgttacgccgagctctaccggcgccagcagcagcagcagcaacagcagccgccccaccaccagcaccagcacctcgcttacgcggcgcccggaatgccggccagcgcggccgacttcggcccgctccaacttggcggcggcggggacgcggaggcgcgcgagccggttgcccggcaccagctgcaccagctccaccagcttcaccagctgcacctccagcagcagctgcaccaacaccaacacccggcgcccaggggctgcacggcggcggcgccggtggcggtggccggggcgcccgcgggctgcgcgggggcgctctcggagctgcccgggtgcgctgcgctccagccgccgcacggcgcgccccaccgcgggcagcacttggagccgctgcagccgggtcccgcgccgctgccgccgcccgcgcccgccgcgctctgcccgcgggaccctcgcgtcccggccgcttgctctgcgccctcggtgcttccgggggctgccccttcgaccgtcgctgcctcctctccgcccgcctctacagccccttcctcatccactggcttctaccggggcgcgtacccggccccctcggacttcggcgtgcactgcagcagccagaccaccgtgctggacctggacactcacgtggtgaccacggtagaaaacggctacttgcaccaggactgctgcgcctccgcccactgcccctgctgtggccagggcgctccgggacccggtctggcgtccgccgctggttgcaagcgcaagtattaccctggccaggaggaagaggacgacgaagaggacgcgggcgacctgggagccgagccccccgggggtaccccattcgccccctgtaagcgcgcccgcttcgaggacttctgcccggactcgtccccggacgcgtccaacatctcaaacttgatctccatctttggctcgggcttctcggggctggtgagccgacagccggactcctcggagcagccgccgccgctcaacgggcagctgtgcgccaagcaggcgctcgccagcctcggcgcctggactcgagccattgtcgccttctagggacccccgagggcatggggaccctgggccccgcggggctggggccagacaaagactcggccaaggggcgatagggagcgagcgggtcctggccactcggggctgagctggggacgagcagaggctgatgttttataaattgtaaaataaaaaaaaaaaatctaaaaatcttggactttatttttgcagagaaaaagcgcctatttaagtatgctttgtgtttctcttactcttttccccccctttttattgtactgattgcagtggtgtttagcgaggagcctgccacgtgaggcagggctgctgcccggaggaggtgcccggccgccgggggcgaggctgggtgcccgggcgcgccaggggcgcaacgcaggagggcgcggggccccggacgccgattctaatcagttgtcaagagccgggaagcccgacgttccgctctcctgagtgcctttctggggtgaggaacgggtcttgtgaaaccctgagcaaaaaaacaaaggcaaactctatctccaaaagggacgtttcggtcacatttcctctctgggggcggcctccaaagttctcaaaatgagaaggcagaaatgaaaacacttcaacttttttttcttttcttgccgggttgggcgtctagaacccctcctctccccgcccctctggctccgtcctcctccccctcctccacccgcctcgcggactcgggcgggggggggggggggcaccctgacactctcagcacagttgggaaaggggtggagggcgagcagggcgaactacatttcccatcatgcccagcactgcggtcctcactaaacaaaaaaggaagtcgattccttcacctggatccccgggggagggaggggcccggaccgccgactgagctggagacttttcgcttagctcctggtcagctctagtgattgtgcgcgcttctaagttaccttgtgctttagccttcggtcccatctcatgaaaccagcattgttccagcctgtgagaaccctgtctccgtcctcagcgcgatagattttgtttaatttaaaagccttttgttgtgagaaggtgaggttcgcttagtgcccctctgggtctctgccatcaacaccccgaggactccaaaacaagttgccaaccttgccttcccggcccttcttcctcctgtatttttgtgcataatgaattgtatctcaccgggtaaaactgttcaggttatttgtttaaatttataatcttaataaaaagtcaattgtagagtgtatggtggttcctttgctttcacatgggcttagaggtgagaaaatctaaacacacacacacacacacacacacacgcaagcaaacaccagctttccagggccatggtggaaacccatggatacccccatatcCGCAGGGCCAGGGTTGGAATTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCTGGGGATGGAGCTAGGGTTGGGAGTCAGTATGTGTGCGTTCTGGGGATGGAGGCCGGTGCTACAAGGTCTTCTCCATAAGCCCTCCTCACCAGCGGGTCTTGCCCCCTTGGGAACCCGCTGCCCAGCAGGAAGCTGAGCCAGCCTCCAACATATTTCCATG

Mutation details

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44453 GAC GTT CCG CTC TCC TGA GT Wild type Forward A
44454 GAG GAA ATG TGA CCG AAA CG Wild type Reverse A
44455 Fluorophore-1 CCT TTC TGG GGT GAG GAA CG Quencher-1 WT Probe
44456 CAC ACA CAC ACA ATT CCA ACC Mutant Reverse A
44457 CAC GTC CAG GAA AGC TAC GA Mutant Forward A
44458 Fluorophore-2 AGA GAG ACT ACC TCG GAG GCT CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44453 0.40 uM
44454 0.40 uM
44456 0.40 uM
44457 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.