Protocol 37171: Probe Assay - Eva1a<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

>chr6:82093116+82093212 97bp TGTGGTCACAACTAAGTCTCTATGG GGGACCCTTTTAAATCCTTAATCC

Mut = 102 bp

Wt = 97 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

TCTCCTTTGTCTCTTTTTGAGACAGTCTCACTCTGGCCTGCATGTCCCAGCAATCCTCCTGTTTCAGTCTCTCAAATGCTGGGATTGTTTATAGGCACAAATCACGATAGCTGGCCTAATAATGCTGTTTGGAAACGTTATGAAATTTGTTCCTGGGAAGGCTGGTCTTGGTTGCCTTTAAGTTCGGGTCCCGGTGAGAGTATGGATCTGTGTCCCTGAccttacatagagacatgactcctgtctgatgccttctccttctctccctgcagaaaaccctgagcgagccgctctgtactttgtctctggagtgtgcattggcctgttcctgaccttggccgccttggtgatgaggatctcctgccacacggactgccggcgaggtccccggaggaggtgcttgcaggaccgcgagtgcagcgacagcagcgacagcgaggatggtagcgaggacacagcctctgacctctcggtgcgcagacaccgccgcttcgagaggactttgaataagaacgtgttcacctctgcagaggagctggagcgcgcacagcgcttggaggagcgcgaacgaatcatcagggagatctggatgaacgggcagcctgaggtgccgggcaccaggagcctgaaccgatactactagggctcagtgtgcaccccagaacctctgcagatcgccgggatgagagtgggttctgcaggcaaggacgatgtgcaagagctgaaaccagctctgttaatgtggctggagagtgcatagcgccagccagcctgtttccccaggagaatggggatttgctttagatccttgcctctggagaaatagacaacccaggcagtggcatctactctttcaggtcttgacagccacctgggcacaccagcccaatgctttcaggccttggaaaggtgctgaggaattcctctccaaccatgtgacagcgatgggacaagctgaaagtccttccgaagcgctcagaagtttgggcagacttatcctgtgctattattcctttataaaaggaaaaaaaaatctcatagctggagagctggggcatcagggtgaccgtaacccagaaaatgtaattttctaaaggactccgtgaagaggaaatggttttgattgagatagcatgatccatgctaattatttaaagctaatttttaaaacagaaaaaaaatcttgacaacctctgcattttaactgacacctcagggattaaaatcctatcttttagtctaggtcccacctcattcatgaagatgaatagaacttactgtattatgagatgtattggagacttaggatacatcaataacctcagaggataaagggttttattttttagtactctataaactatgtattgacatgtgtatttgaaaatgctgcataaaaatagaaaccgtgtgtcctttccttttggagagaaaagtctcatgacttcagagactcatgatatttaaataatattttgtttaaaaaaatagtttggatttccaagaatggaagccagaggtgagaattaaatccaaaattagaatggtatcgaaaataaacattccatgaaaatcttcttttgcttcgtgtctgtgtggtcacaactaagtctctatggttaacgaccttagttacaggcaagtatgacctctgcaggacTCCGGAAGGATTAAGGATTTAAAAGGGTCCCTGGAGTTAAGGGTTCGGGTTGTAGGTTGTAGGTTGTAGTTGAGTTATTGTTAAGCTGATAGAGGAACTGAAGGCTTAAACCTGAGGTAAGAGATCAGATAGAGACCCCACCCTCAGCCCCACCCCCTCTGTGGTGAGGAGAAATTTGTTGGACTCG

This mutation is a 1441 bp deletion beginning at Chromosome 6 position 82,091,741 bp and ending after 82,093,181 bp (GRCm38/mm10). There is a 1 bp (A) insertion at the deletion site. 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50719 GGG ACC CTT TTA AAT CCT TAA TCC Common A
50720 TGT GGT CAC AAC TAA GTC TCT ATG G Wild type Forward A
50721 TTC CTG GGA AGG CTG GTC T Mutant Forward A
50722 Fluorophore-1 ACC TTA GTT ACA GGC AAG TAT GAC CTC T Quencher-1 WT Probe
50723 Fluorophore-2 TGG ATC TGT GTC CCT GAA TCC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50719 0.40 uM
50720 0.40 uM
50721 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.