Protocol 37902: Probe Assay - Zfhx4<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

 

>chr3:5241675+5241759 85bp TGACAGGCTGGATGAAATGA CCATTTTCCTGCCTTGAGATAG

Mut= 85 bp

Wt= 85 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

TTTTAGATAGTCAAGTTTACAGGAGCTGTGAATTAGTGATGGGCTATTAGTGAGCTGTGTCATTATTTAATAAAAATGGCTTCTCTCACCTTATTTTTTATCCAGGTCCCTGACAGGCTGGATGAAATGAGATCCCCATGTAGCAATTGCcatggaaacctgtgactctcctcctatctcaaggcaggaaaatgggcagagcacatcaaagctatgtggaatgacacaacttgataatgaggtgccagagaaagttgcagggatagagcctgacagggaaaacagctcctcacatgacaacctgaaaacggatgagcgcaaaagtgaagtcttgctgggtttcagcattgagaatgcagctgccactcaggtcacctcagcaaaggaaataccctgcaacgaatgtgccacttcttttcccagtttacagaaatatatggaacaccactgccctaatgcccgccttcctgtcctgaaggatgacgagagtgagaccagcgagttagaggacagtgatgtggaaaacttaacaggggagatagtttaccagcctgatggatcagcgtatataattgaggactccaaagaaagtggacagaatgcacagactggggcaaacagcaaactcttttcaacagcaatgtttctggactccctggcatctgctggagagaagagtgatcagtcttctacggcccctgtgtcattctacccacagatcatcaacacttttcatattgcttcatccctcgggaaaccatttacagccgatccggctttcccaaatacctcagcattagcaggagttggtcctgtgttgcacagtttccgtgtctatgatctccgacacaagagagagaaagactatctaaccagtgatggctcagccaaaaactcctgtgtgtccaaagatgtccctaacaatgtggacttgtccaaattcgatggttgtgttagtgatgggaagaggaaacctgtcttaatgtgtttcttgtgcaaattgtcttttggctatatcaggtcatttgtaacccatgctgtacatgatcatcggatgactctcaatgatgaggagcagaggctcctcagtaataaatgcgtctccgccataatacaggggattggcaaagacaaagaacctcttataagctttctggaaccaaaaaaatccacttctgtttatcccaatttttctactacaaacctcataggacctgatccaaccttccgcggtttatggagcgcttttcatgttgaaaacggtgactctttgcaggctggctttgccttcttgaagggaagtgcaagcccttccagctcagcagaacagccgctggggatcacccacatgccaaaggctgaagtgaacctgggggggctgtctagtttagtagtgaacaccccaattacctccgtctccctcagccacttatcatctgagtctagcaagatgtcagagagcaaagaccaagagaacaactgtgaaaggccaaaagaaagtaccatcttacacccgaatgtggggtgccctgtcaaaagtgaacccactgagccgggagatgaggatgaagaggatgcatattccaatgaactcgatgacgaggaagtattaggtgaactcacagatagtattggtaataaagatttccctctcttaaaccaaagcatttctcctttatcatccagtgtgctaaaatttattgaaaaaggtacctcgtcctcctcggggactattgctgaagacactgagaagaaaaaacaggccgccgctgctggaagaagcaatggcaacgttactaatagctatagcattggtggcaaggactttgcagacggaagtatcagtagagatggcaccacagcagctcctagtgaaacgacccatggagacgaggacagttcaacaacacaccagcatggcttcaccccgagcacacctggtacaccaggtcctggaggagatggctccccaggcaatggcattgagtgtccaaaatgtgacacggttttggggtcttcaaggtctcttggtggtcacatgactatgatgcactcaaggaactcatgtaaaaccctgaaatgtcccaaatgtaactggcactacaaatatcagcagactctggaggcccatatgaaggagaagcacccggagccaggtggctcttgtgtttattgtaagactggacaacctcatcccaggcttgcccggggagagagttacacgtgtggctataaacccttccgttgtgaggtttgtaactactctaccactaccaaaggcaacctcagtattcatatgcagtcagacaagcacctgaacaatgttcagaatcttcaaaatggcaatggcgagcaggtgtttggccactctgctccaacccctaacactagcctcagtggctgtggaacaccctccccatccaaacccaaacagaaacccacctggcggtgtgaagtctgtgactatgagaccaacgtggcgaggaacctccggattcacatgaccagtgagaagcacatgcacaacatgatgcttctgcagcagaacatgaaacagattcagcacaatctgcacctgggcctcgccccggccgaggccgagctctaccagtactacctggctcagaacatcggcctgacagggatgaagctggaaaatcctgccgagactcagctcctgcttaaccctttccagttcgactccgccacagcagcggctctggcacCAGGACTTGGTTAGTACTTTCTGTTCTGCACTATTGTCCGTTTTGAATATTTTCCACTTTGATCTCTTGTAGGAGGGCACTGACAAACTGGGACTGTTCGCCAGAAAGGGCTTTCTCTTAAACTGCGTAGGCAAACTGCAAACAGGCACATTGCTTTAAAGCTAAGTTAAATCTAGAGAGTGAAGAGGTGAGAAATTAGTGACAGCA

This mutation is a 2576 bp deletion beginning at Chromosome 3 position 5,241,715 bp and ending after 5,244,290 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52460 TGA CAG GCT GGA TGA AAT GA Common A
52461 CCA TTT TCC TGC CTT GAG ATA G Wild type Reverse A
52462 CAA AAC GGA CAA TAG TGC AGA Mutant Reverse A
52463 Fluorophore-1 CAT GGA AAC CTG TGA CTC TCC T Quencher-1 WT Probe
52464 Fluorophore-2 AGC AAT TGC CAG GAC TTG G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52460 0.40 uM
52461 0.40 uM
52462 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.