Protocol 37519: Probe Assay - Znhit2<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

>chr19:6062321+6062431 111bp TGTGACAGCTGAGGAGATGG CCAGTTCAGCCAGGGAGTTC

Mut = 120 bp

Wt = 111 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

AAAGAAGTACTGGAGACACGAAAGTTACCGAGAAGTTTCGGAAATGTGCGGAAAGGAGGCCACGCCCGCCAACCACCCCCACACATAAAGGCGGAAATAAccaaagaagcgccggaagtggcggagctgatacgggacaggcggcaccagtttccatggagcccgctgggctctgcggcttctgcccggccggggaagcgctgcctgcgcgctacacctgcccgcgctgcaatgctccctactgctcgctgcgctgctaccgggcgcacggcgcctgcgccgaagacttttaccgcgaccaagtgctgcgagagctccgtggccgaagcgcttcgcccagccgtctggcgggcgcgttacgccggctgcgcgaacaacgcgaggccgaggatgagcccgaggaagcaggcctcgggcccggggcgcggccgggcggcctttctggactttgggaacgtttaacgccggctgagaaggcggcgttcgagcggctgctgagccgtggcgaagccggacgccttctgcctccgtggcggccctggtggtggggccgcggcaccggcccacgtttgctggaggagctggatcatgccgcgaacagagatcttgcggagccggagcccgcccctgcgaggaccgcgctgcaatctggggatgatgccgctgccgcagagccatttgctgaagactcctgtgccgccagaccgctagcattgcctgctcgcatcccagcactggccagtctgagccgcagcccggcatcgccgctggtgcgcttccagctgcccaacgtgctgttcgcctacgctcatactctcgccctgtatcatggaggggacgacgacgcgctactctctgacttctgtgccaccctgctcgatgtttctggggccctgggagcccagcaagtctttggctctacagaggaagccctgcaggccgcagcccacgtactggaagcgggcgagcacccacctgggcccctgggtacgcggggtgctatgcaagaagttgcccgaatcctgctgggcgagggtccagtcaatcagaaaggctacactctgacagcactggggcacctagctcagaccctgggacgagcccggaagcaggctgtgattggtggagagcgagatcgactttaccgggctcggaagaagtgccagttcctgctggcttggaccaacgaaaacgaggctgccctcacacccctggcgctagactgtgccagagcccaccgagcccatgctgtgacagctgaggagatggcaacgcttactggagagttagaacggctttggggaggcccagtgccacccactccaaggactctcattgaggaactccctggctgaactgggtatccatgtcattaataaagatgtgtctggtctgcccgaatgtcagcagtgccttttccgtaaagatagtgccctgtcctaggcttctcttgaagttactgtgtagttgaggatgaccctggaactttgatctgtcacccacctggacagtgagaggttacaggggttaaccaccttgctaatgtacacagtgctgaatagccgaggcgtcctgtgtgctacacaagcactctataactgaactacatcacatcccccaggcctgaggtgcccagttgtctctcctcgtcagacttttggacCCACAAACCACTTCTAAGCTTTCCTTAGTCAACTAGTCACCCCCAAGCCTCAAGTCACTGGCCCCAGGATAGAAGAAAAACACAGGAAAGGTTGAAAACATTTATTCAACAGGGAAAAATACTTTCCTTCTGGAAGCAGTGGCTTCCTCTTGCTTTCAACCT

This mutation is a 1563 bp deletion beginning at Chromosome 19 position 6,061,172 bp and ending after 6,062,734 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51563 TGT GAC AGC TGA GGA GAT GG Wild type Forward A
51564 CCA GTT CAG CCA GGG AGT TC Wild type Reverse A
51565 AAC CAC CCC CAC ACA TAA AG Mutant Forward A
51566 CTT TCC TGT GTT TTT CTT CTA TCC Mutant Reverse A
51567 Fluorophore-1 CTT TGG GGA GGC CCA GT Quencher-1 WT Probe
51568 Fluorophore-2 AAC CAC AAA CCA CTT CTA AGC TTT C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51563 0.40 uM
51564 0.40 uM
51565 0.40 uM
51566 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.