Protocol 37919: Probe Assay - Zfp729b<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

>chr13:67593824-67593920 97bp CACAGAATGCAGAGAATATGGA TTCACTCCTGTAGGGCTTCAC

Mut= 106 bp

Wt= 97 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

GGCTTCATTGAATACAGGTGTAAAAAAATGAATGTAGCTTATCATTTATAAAGACAAGTGCAAGTAAGACAAGACAAGTGAGAAATGTTGGTTTTTAAAGAGAAACATTATAAATTTACAGAATTTTACTCTGCTTTTATTAATTTCTCAATCGTTTCTCCACAGAATGCAGAGAATATGGAAaagtttttgagtggtccaaagtgtttcaaaatcaccagataattgacttagaagtgaagccctacaggagtgaagaacgtgccaagtccttccatattccatcattaagttctgaagagagcacaattcatgcaggagaaaaaccttacaaatgtgaagtttgtggcaaggccttctgcattccattattactttctaaacacaagataattcataaaggagagaatctctacaagtgtgaagtgtgtggcaaggccttccaacatccatcaagactttccagacataagaagattcattcagaagagaaaccatacaagtgtgaagtatgtggaaaagccttccactttccatcattacttttggtacacaagagagttcatacaggagaaaaaccatacaagtgtgaagtatgtggcaaggcctttcattatccatcaatactctctaaacacaagcgaattcatacaggagagaaaccatacaagtgtgaagaatgtggcaaggccttccacatttcatcatttctttctaaacacaaaataattcatagaggagaaaaaccctacaagtgtgaggtatgtggcaaggccttccattatccatcaagactttccaaccataagaaaatccattcagtagagaaaccatacaagtgtgaaatatgtgggaaggccttccgcattttatcactcctttctaagcacaagataattcatacagaagagaatccttacaagtgtgaagtatgtggcaaggctttcgattatccctcaagactttctacccatgcaaaaatgcatacaggagagaaaccatacaagtgtgaagtatgtgaaaaggctttccgttctctgtcatcactttctaaacacaggagaattcatatgggagataattattataacagtgaattatgtagcaatgccttcgtttatccttcaagactttctaaacataagaaaatttgtagaggagagaagccctacaaatgtgaagtatgtggcaaggccttccatgtttcatcactgctttctaaacacaggacaattcatacaggagaaaaactctataagtgtgaagtatgtgggaaggccttctattatccatcaagactttccacgcataagagaattcatacaggagaaaagccatacaagtgtgaagtatgtggaaaggcgttctgttttccaccatcactatctaaacacaagagaattcacacaggagagaaaccatacaagtgtaaagaatgtgggaaggccttccgatttccatcatcactatctgcacacaagaaaattcatacgggagaaaaaccctacaagtgtgaagtttgtggcaaggccttccattatccatcattactttctaaacacaagataattcatacaggagaaaaaccctataagtgtgacatatgtgaccaggcatttcatgttccatcaaaactttctcatcacaagataattcatacaggagaaaaaccctacaagtgtgaagtatgtggcaaggccttccattattcatcactactttctaaacacaagattattcatacaggaaaaaaaccctacaagtgtgaagtgtgtgacaaggccttccattatccatcaagactttttagccatacaaaaatgcatactggagagaaaccatacaagtgtgaagtgtgtggaaaggccttctgttctccatcatcactttctaaacacaagagaattcataaagttgaaaaagcctacagttgtgagatttgtggaaaaaccttctgcATTCCATTATTACTTTCTAAACACAAGATAATTCATACAGGTGAGAATCACTATAACAGTGAGGTATGTAGCAAGTTCTTTGTTTATCCTTCAAGACTTCCTAAACATAAGAAAATTTATAAAAGAGAGAAACCATACAATTGTGATGTATGTGGGAAGGCCTTCGATTATCCATCAAGACTTTC

 

This mutation is a 1775 bp deletion beginning at Chromosome 13 position 67,592,123 bp and ending after 67,593,897 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52506 CAC AGA ATG CAG AGA ATA TGG A Common A
52507 TTC ACT CCT GTA GGG CTT CAC Wild type Reverse A
52508 ACA AAG AAC TTG CTA CAT ACC TCA C Mutant Reverse A
52509 Fluorophore-1 AGT TTT TGA GTG GTC CAA AGT GT Quencher-1 WT Probe
52510 Fluorophore-2 ACA CAA GAT AAT TCA TAC AGG TGA GAA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52506 0.40 uM
52507 0.40 uM
52508 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.