Protocol 36347: Probe Assay - Znhit1<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

>chr5:136982022-136982119 98bp TCTTCTTGTAGCCTCCATTG CAGCAGCTAAGAAAGGCAGA

Mut= 116 bp

Wt= 98 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GCCCTCTCTCCAGCTGCAATTTATTGTGTCTCCAGTTATTGCTGGGACTGGGAGGACTTAGAGGCAGCATAAAGGGCCGAAACAAGCTTGCCAAGCTGGAAGAAAGCAGGGTGAAGATCTGGGAATTCCTCACAGCATAATGAAGGGGCCGAGGCGAGGAGACTGGGGCTCTAGGCAACGGCAGTGCAACTTTCCTCCTGTCTTGACCCCGgtaactctctttgccccatctagttcgctcccaggaccctggacagcggcgggtgctggacagggcagccaggcagcggcggatcaaccgacagctggaggcattggagaatgacaacttccaggatgacccccatgccggcctcccccagctgggcaagaggctacctcagtttgatgatgatgcagacacaggtgagatgggggttagggaggaggagcgagacagtgccgtgaagctccttgtcacccgtaactgcagatgatgccaatgagcgcagatgggaattgcatctttattgtgtttttaatttcctcattttgtgtgcatgcatatgtgtgtctgttcattcctgcacacgcttgcctgtggagatcagaaattaacttacaggagttcgttttctccttccagagttgaaattctggttactcaggttgctgagttcagcagcaggttcccttaagcaccaagccatcttagcagcccctgtttctttaaaactgcaacttattcagagagcaggcaatctgagaaatttattgatattaattaatatcaacttattgatattctaaagatccatcttctagcctgggggagccattgctcacatgtgcacgtgtgtttacaaatacacacactcacacacacacacatacacacatgacacaatgacacatacctttaagcccagcactagggacacagaggcaggtggatttctgtgggtcagcctggtcttcaattggagttccagaacagctagagttacacagaaaaaccctttctcaaacaaacaaacaaacaaaccatgttcagtcagcaggttatagacagagacaaaaggctgtcaatcatctggattggttgccatttccccttccctctgtgtcaggtgtcagccacatctctgagactagcgatggccgtgacctttgttatcatcctgtgtacttgttccttactctgctcatgtctgggctgggctccggcacttcttgaaccactgagtcaaattagtgtctgctggtgtcatctttgtactccaaggccatctgacagggagggggtggttagctcagcaaaccaaccaggaacagctgtatacgctaagctagaaacaccatgtatgtgcctgttgtctggtctgtttatagtagggagggttttgtttcttgagagagggtctctctatgtagaccaggctgttctggaactcacagagatccgcctgcctctacctcccaggtgctgggatcagcggtgtgcgccacgcccggcaagatgtttttaaactttaaataatctctcttgtttcatgtgtggcatggaggtcagaggacaatgtttgggagtagcttttctccttgtactgagggccagggactgaactcaggatggcagccttggcagccagcgccttttcccgccatcttgccggctcaagtgtgtcatttatacaaatgtaggcagagagggacagaccacataaaatatccctgcccttgggctggaggaaagggaaggcgtcgggactgtgggagggaaggctggcagtagggcggtgacttgggaattacttctggctttgccagtgcctggctcctgtgttgggagcatctgcctttcctgtctgtgtctctgtgcctcagtttcttcatatgtgtgggataaaaaagatggagcaatcaggtatagcgacccagcacacagaaagtttgaagccagagaactgccaagaatttaaggtcagcctggtctatatagtgagtttgaagtgggcttggggtaggtagtgagaccttgtatcaaagcaacaacaacaagactgtgtaatctgagcacctgagaggtgggaacagggagatcgatcaaagatcaccgtttatctacacagtgagttcatggccaacctgggcttcatcagctcctgtccccaaaataaaaagatggagtaggttggacttttaagtagctgacggtcgaccaggattgcagatatcagtcagcgttagtgcagatgtaggcagggagggacagaccccgattgtggtgtggataggcctctccaacaggactgtcacagccactgtccagcaactcagtgtccttcatcccccaggaaagaaaaagaagaaaactcgaggtgaccactttaaacttcgcttccggaaaaacttccaggctttgctggaggagcaggtgagagaaggctcagtctgaggttgggaccctagagagcacggggggggggggggggtgggggggggggtgggggggggggggtggtgagcaaggtcctgctcttcagcgccccttgtggacatcagctgcatggcggactggagaattccggatgcctgacccttcatgtccttgcagaacctgagtgcatctgagggccccaactacttgacagcctgtgccggccccccatctcggccccagcgtcccttctgtgctgtgtgtggcttcccatccccgtatacctgtgtgagctgtggtgcccggtactgcaccgtgcgctgcctgggtacccatcaggagaccaggtgagcccgagcctgacccagccatgcctacagtcttattccatgtagcatcccaggacctccctccttctccaatgggcttctctctctctttctgcaggtgtctgaagtggaccgtgtaaacctggcatcagggagagaaagtacccccacccttcccaacccaccccagctcattcacaaaggacagagagagggagaaactacccattcacccagcaaagctgtgccctgccgaccaaggaaggccacagggacccaggactgttgtgggggtggtccatccacagagtgctgtgataataaacggcctggtgaaaggaagcctgcctgctcattctggggtatttggaggtgaaggtcagggttgactgtcaggccagcatagactctgcctttcttcttgtagcctccattgccggccctctgtgtcatGGGAGGGTTGTGTCCCATTCTCCAGCTACTCTCAAGTCATCTCTGCCTTTCTTAGCTGCTGCTTTACTTTGGTTTTTGAAACAAAATTTTGTATAGCCTAGGCTAGCCTTGAATTTACTATATGTATAGTCATTAGGCTGAACTCCTAATCTTTCTGGCTCTGCCTCTGCCTCTGCCTCTTAAGCGATGTTTTTTAAATAGTTTTTCTTTCCATGTATCTG

This mutation is a 2990 bp deletion beginning at Chromosome 5 position 136,982,083 bp and ending after 136,985,072 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48482 TCT TCT TGT AGC CTC CAT TG Wild type Forward A
48483 CAG CAG CTA AGA AAG GCA GA Common A
48484 AGG AGA CTG GGG CTC TAG G Mutant Forward A
48485 Fluorophore-1 CCG GCC CTC TGT GTC A Quencher-1 WT Probe
48486 Fluorophore-2 CTC CTG TCT TGA CCC CGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48482 0.40 uM
48483 0.40 uM
48484 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.