Protocol 35546: Probe Assay - Tifa<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

>chr3:127796380+127796470 91bp CCCCTACACCATTGTACTGTGA GCCCTTTCCTCTTGCTTTGT

Mutant=  107 bp

Wild Type = 91 bp

Sequence

Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion):

 GTCCAGGCTGGGCTTGAACTAACGATCTTTCTGCCTTAACTTCTCACCACCACATACTTGATTTAATTTTTAATTACAACCATCATATTTTTTTGTTTACATCACTATTTAAATGAGAAAGGAGTGAGACTTCCCTCTGCTTCCCCTACACCATTGTACTGTGAGCAATCTCATAACcgtccacattataagactgggcaaaggagaaatgaaacaaagcaagaggaaagggctagtattgttcttactataggatcccctaccgcattttgagcacactctttgagagggcagtacaggctgaactaaccgcatgctttgaccattgcaggccggggagcgtcatgtccacctttgaagacgctgatacagaggagacggtcacttgtctccagatgaccatttaccatcctggccaacaaagtgggatatttaaatcaataaggttttgcagcaaagagaagtttccttccattgaagtggtgaaatttggacgcaattccaacatgtgccagtatacgtttcaggacaaacaggtgtcccgaattcagtttgttttacagccgtttaaacagttcaacagctccgttctctcgtttgaaataaaaaacatgagcaagaaaaccagtttgatggtagacaaccaggagctcggctacctcaataaaatggacctgccttacaagtgtatgctcaggttcggagagtatcagttcctgttgcagaaggaagacggagagtcggtggaatcttttgagactcaatttatcatgtcttcaagacctctcttgcaagaaaacaactggccaacacagaatcccataccagaggatgggatgtattcttcatacttcacccacagaagttctccttcagaaatggatgaaaacgaactgtgaagagggtccaactggagacacattgaaggatgaggacacatgggtcggatgtcaagagacatcctacttccgagtttgtgagtgtagcgtagcgcggctgtcctcatgctgactttcgttttggtaatagcatttggaagtctctagactgtgttaatcatcaacttagtcaactgagtttcggctctacaaagaattaagtgtacatctgtaagggttggtgcatcagacacgtcttctgggtaatgaggtcacccttgttgcttttctgcatgatgttacccccatgctttgtcttggtggcagccatctcttggcccggtcacatcatttcgtagcagcctttgtttttcaggtttagagctcgggcagattgctcactggtgtctgtggcgtgctagcgcttgtagaactagagtcctggaataagttctagagtgctgagtcactgagtcaccatggcttccttatggaaagacttgggaaatagctccttgattttctttctgtggaacggtagtgtcgctttcctatatgtaggacctacaacaaacatttaaagaacactgagatgaagatggttttcttacaatattgaaagtgaattttatgtatctcacagatttaaaaatggcagaaatcaaaacttttaacagcctctttgcacatgataaagccggagcccagttccttagttgcttctttggaacttcttaaaggaaaacatgtattcttaaaggaaaacatctattcttaggctgccctatagaagtcagtacctgtgaatatttatattaaatgcttaattatttctaaaattttagtttcacataaagttgtatttatttaaaagattctcattcacttcattttggctagattaagatgaatgttagtgaacattatgtaaaagaggatgaaagccattaagttaagataaattctagcattactagtaagtaaggcaccctgtatagcttcctctgtaaatgaaatttaatgctgtaacaggtacaggattttgggtaggggaggaggtcaggtgggggaagttagccacattcatattttgtttttgtttttgtttttgtttttgtttttgttttccaacaatagcttgctttgaagctcaggctggcttggaactcttgatcctcatacatcggccccctgaatgctgtgcctagcttaatgtaactgtatttctgcaacagccctttgaaattatttctaataaactgtttggcctagtaccatgtgaatgttatctgcagtatgcctcttgctgcagatcacCAGTCAGTCACCCCTTGCCTATAAAATAAGACATCAGCTGCTGCCTCAGCAGGGATTGCTGCTATAGGCAACCCTTTATGTGCTAAGTGTCCAGGTGAGTATTTCATTTCTTGCCTTGTTTTCCTGCTGACTCCCGTGGTAAGCTGTTGTGTGTAGCTTTTCACTATCCTAGCTATGCTCTGGGAATCAGACTGAACTACCAGCCCCCTCCCAGCTTCCCTTGAATCCATGGATA

This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences GCCCAGTCTTATAATGTGGA and GACTGGTGATCTGCAGCAAG, which resulted in a 2018 bp deletion beginning at Chromosome 3 position 127,796,415 bp and ending after 127,798,432 bp (GRCm38/mm10). This mutation deletes ENSMUSE00001352154 (exon 3) and 197 bp of flanking intronic sequence including the splice acceptor and start of translation and is predicted to result in a null allele.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46353 CCC CTA CAC CAT TGT ACT GTG A Common A
46354 GCC CTT TCC TCT TGC TTT GT Wild type Reverse A
46355 GTT GCC TAT AGC AGC AAT CC Mutant Reverse A
46356 Fluorophore-1 CCA CAT TAT AAG ACT GGG CAA AG Quencher-1 WT Probe
46357 Fluorophore-2 CAT AAC CAG TCA GTC ACC CCT T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46353 0.40 uM
46354 0.40 uM
46355 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.