Protocol 35487: Probe Assay - Nol4<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

>chr18:22823048-22823183 136bp ATTCACAAAATGTTATGTCAGGGTTA CCCAGGGAACTGATTCATAAGT

Mut= 144 bp

Wt= 136 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TATTGCGGGATCCTCCGGTAGTACTATGTCTAATTTTCTGAGGAACCGCCAGACTGATTTCCAGAGTGGTTGTACAAGCTTGCAATCCCACCAACAATGGAGGAGTGTTCCTCTTTCTCCACATCCTCGCCAGCATCTGCTGTCACCTGAATTTTTGATCTTAGccattctgactggggaggaataatcacatcaatttgtaaaccaattggtaattgacttattcactttgttagattgacttttctttcaggttgcttttgatataagccattttcttttcttattcatgtttgttctttagtgtgtttactagattcaaaaataacaattgctgcttcataacaaggttaccacttagttaaattacctatatgaataaaaccagagacacattgcttcccatctttcagaacttagtcaacactagttgtagatgatctagcatattacttaaactgtcaatctttcttcctaacagatgactctgctgcagagagctctaatggcaatgagactctggggcacagctcagctgcttcagggggagcacatggcagggagcccgaggactccagcagtgatggcaaaaccgggctggagcaggaagagcagccactgaacctgagtgacagccccagctctgcacagctcacttcagagttcaggatagatgaccaaggcagtgatgggaaaaacaagtataagaatcttctaatctctgacctcaagatggaacgagaagcgagagaaaatggaagcaaggtaaggcttcactacattgctgagtgggctttatgttggtcatgaagattaagttggtaacacatgaacattactaataaatgcataaccatgcattatgtatattcacaaaatgttatgtcagggttaatagagccaagtgaaatttatctcaaagctgtgGTTATTATTTGATATAGAGAATTGTTTGATTTAATGCATTTCATTTCTATAATCTACTTATGAATCAGTTCCCTGGGAATTTTCCAAGTGCAAATGTAAAAGGTTTGGTCTCAGTTCACAATTTTGAACAATAAAAGGATCACTCATTTCTCTA

This mutation is a 763 bp deletion beginning at Chromosome 18 position 22,823,125 bp and ending after 22,823,887 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46197 ATT CAC AAA ATG TTA TGT CAG GGT TA Wild type Forward A
46198 CCC AGG GAA CTG ATT CAT AAG T Common A
46199 TGG AGG AGT GTT CCT CTT TCT C Mutant Forward A
46200 Fluorophore-1 GCC AAG TGA AAT TTA TCT CAA AGC Quencher-1 WT Probe
46201 Fluorophore-2 TGT CAC CTG AAT TTT TGA TCT TAG GTT A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46197 0.40 uM
46198 0.40 uM
46199 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.