Protocol 31870: Probe Assay - Ppfia3<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

>chr7:45354619-45354713 95bp CCCTCCTCCTATTCCAGGTG AAGGGATGAGAGTTGAGAAGGA

Mutant=  98 bp

Wild Type = 95 bp

Sequence

Wt Sequence: gggcacagtgaggatatctcaggggaggggctaaaatgggggcggggcttagatgaggagccgggttgagtgaGACATGAAGGGTGGGGCTCAACTGAGGATCAGAGTAGTGGGTGGGACTGGTTTGGTTGAGATGAGGGATGGGTTAAGGAGGGACTAAACAGGAATCAAGATGAAGCAAGGCTTAGATGAGGGGCATGATTTCGAACAAGGGGAATGATCTCAGAAAGACCCGCTGGACAGTCTCTGCCCTGTTTCCCTTTGTTCAGGCCCGGCAGAGGGAGAAAATGAACGATGACCACAATAAGCGACTGTCAGAGACGGTGGACAAGCTGCTAAGTGAATCCAACGAACGGTTACAGCTTCACCTCAAAGAACGTATGGGGGCACTAGAGGAGAAGGTGCGCCCCCTCCCACCCCTGCCCCAGACCTTTGTCAACCAGGGGTGGCTCCAAATGCCGTCCTAGGGCCCATACATCTTCAGATCTGTCATTTGAGACCTCCATACCCACAAACTCTGCTCTGTGACTGAAGGCACCATTTTGCTTGGCCATTCCAAGAGCGGAGTTTTCCTAGACCACAACCCCTGCAGCCTGCCTTTGACCTTCCATTTCACCCCTGTCCCCACAGAACTCACTGAGTGAGGAGATTGCTAACATGAAGAAACTTCAAGATGAGCTGCTGCTGAATAAGGTGAGGACCCGGAACTTAATGGGGAGTTATCGAGTGGCCTGTTCCTCATAGCAGTGACATCGAGCCCCATACCTCTGCCTCTCTCCACAGGAGCAGCTCTTGGCTGAGATGGAACGGATGCAGATGGAGATCGACCAGCTCCGGGGAAGGCCGCCCTCCTCCTATTCCAGGTGACAGAAGTGCTTCCTGCCCTTTCTGTCTGTGgcaagctcctcctttccgctcctccttctcaactctcatcccttattagtttatttattttatg

 

Deleted Sequence: GACATGAAGGGTGGGGCTCAACTGAGGATCAGAGTAGTGGGTGGGACTGGTTTGGTTGAGATGAGGGATGGGTTAAGGAGGGACTAAACAGGAATCAAGATGAAGCAAGGCTTAGATGAGGGGCATGATTTCGAACAAGGGGAATGATCTCAGAAAGACCCGCTGGACAGTCTCTGCCCTGTTTCCCTTTGTTCAGGCCCGGCAGAGGGAGAAAATGAACGATGACCACAATAAGCGACTGTCAGAGACGGTGGACAAGCTGCTAAGTGAATCCAACGAACGGTTACAGCTTCACCTCAAAGAACGTATGGGGGCACTAGAGGAGAAGGTGCGCCCCCTCCCACCCCTGCCCCAGACCTTTGTCAACCAGGGGTGGCTCCAAATGCCGTCCTAGGGCCCATACATCTTCAGATCTGTCATTTGAGACCTCCATACCCACAAACTCTGCTCTGTGACTGAAGGCACCATTTTGCTTGGCCATTCCAAGAGCGGAGTTTTCCTAGACCACAACCCCTGCAGCCTGCCTTTGACCTTCCATTTCACCCCTGTCCCCACAGAACTCACTGAGTGAGGAGATTGCTAACATGAAGAAACTTCAAGATGAGCTGCTGCTGAATAAGGTGAGGACCCGGAACTTAATGGGGAGTTATCGAGTGGCCTGTTCCTCATAGCAGTGACATCGAGCCCCATACCTCTGCCTCTCTCCACAGGAGCAGCTCTTGGCTGAGATGGAACGGATGCAGATGGAGATCGACCAGCTCCGGGGAAGGCCGCCCTCCTCCTATTCCAGGTGACAGAAGTGCTTCCTGCCCTTTCTGTCTGTG

824 bp deletion beginning at Chromosome 7 negative strand position 45,355,486 bp and ending after 45,354,663 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37874 CCC TCC TCC TAT TCC AGG TG Wild type Forward A
37875 AAG GGA TGA GAG TTG AGA AGG A Common A
37876 CAG GGG AGG GGC TAA AAT G Mutant Forward A
37877 Fluorophore-1 TGC TTC CTG CCC TTT CTG Quencher-1 WT Probe
37878 Fluorophore-2 CTT AGA TGA GGA GCC GGG TT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37874 0.40 uM
37875 0.40 uM
37876 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.