Protocol 35098: Probe Assay - 4933402J07Rik<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

>chr8:87567771+87567864 94bp AAACTTGGTTTCCCACTTGC AAGGCAGGATAAAGGGCTCTG

Mut= 90 bp

Wt= 94 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CTTTGGGGATCTTGTCACAGATGCCAGGGATACATGCCAGGGTGGTCCAGAGGCCAAGGGACAGTGAAGGGACAAGAGGCTCTAGAGTATTGGTACTAAAACTTGGTTTCCCACTTGCCAGGAATGGCCTCCTATTTAGAGacagtgggtagaacccccacaggatggctgcagagccctttatcctgcctttacagaaaaagaaggaagagacaaagagtacagaagagaaggcaggaaagaaaaacgtaggcgctaagaagaagctagaaaaagtcagccgcaaagtgaccttccacaaggtaacacctctccccctcccccacagctgctcctctactatggtgtctgccacccctctctgaaccataatgacaccctaagagatgagtcttctcacgcgcgcgcacacacacacacacacacacacacacacacacacactccttgcagcttccccaacaacctctgctttcctgcagtccagtttaagggtgagcgtctcacattcctccacccacgtccctccatactccatgttaccagggcccagagaaccatgggaacactcagtcctgtacctacagggtatcccagacctgcctgcgagcatagactgcatgtaccctcatgagacaaggagaaataaaagtgaagtctcagggagccagcatggaagacacttctgctccctgaaactcttcctgggctcagaccaggtcctcgagcaagctggagaaatttctcagtttgggtttagaggatgctgagcaaggtcctctaagcagagcataacttctctcctgctttccaagggctacgtacgtcctgtcaagaagaaggaacccaccatttaccacaagagaaatgaaatagacctaaagagcaagcgtctgagcatgatcactgccaacttcagggatagcaataagaagtctggtaaggaaaatacacaggccctgggataccagaactgacctcccttgtccaatttctaagagataatggaagagctgcagcatccatgctgatccaacctgtgtacataggagccaggagccagcatgcagggttgttagggaatcacccagGGTAGCATTTGACAATCTGGGTATGCTGTGGATAGGAGGGGAGTGGTTGTTGATCTCTACATTGTCAGGAATCCAGCACAGTGTGATACATGCTCCTGAACTATTCCAGTGATGTCCAAATTCTTTAACTCAAACACTAT

This mutation is a 950 bp deletion beginning at Chromosome 8 position 87,567,814 bp and ending after 87,568,763 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45262 AAA CTT GGT TTC CCA CTT GC Common A
45263 AAG GCA GGA TAA AGG GCT CTG Wild type Reverse A
45264 ACC ACT CCC CTC CTA TCC AC Mutant Reverse A
45265 Fluorophore-1 CAG TGG GTA GAA CCC CCA C Quencher-1 WT Probe
45266 Fluorophore-2 AGG GTA GCA TTT GAC AAT CTG G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45262 0.40 uM
45263 0.40 uM
45264 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.