Protocol 36640: Probe Assay - Efhb<em1(IMPC)J>Alternate2
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

>chr17:53452568-53452713 146bp TTTTAGGTACAGCCCAAGT AGCTTCTCAGTCAAACAGGTTGC

 

Mut= 162 bp

Wt= 171 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

TCTGTAGACCGGCTGGCCTCGAACTCAGAAATCGGCCTGACTCTGCCTCACAAACGCTGGGATTAAAGGCGTGCGCCACCACGCCCGACTTTACCGGGATGCATTTCTGCTACTTGTTTGTTTACTATCTTTTCAATTGTCCTCTTGTTTTATTTTAGTAGCATTTGTAGCGTTTTAGGTACAGCCCAAGTTTAAAATACATTCCACTCCttaaccaggctttcttgaccctgaaatgatcaacgctttttttccctctaaggcaggaaaagttaagccagttggatacagagttgcaacctgtttgactgagaagcttcccagggtgagcacgtcccagtcttcctgtttgttcctcaaatatgagttgtagggagactagattctcagctttctttatgaaagggtgcacagatgtaacgtgtctagttttgaatctttactatattttggcgttatctattgatccatattcatccaagaaaatgaaaatgaaaataaacatttttagtttaccaaaaattctaagagtcaatgtatttcagtaggaattatgggatacttgtaacctggatgtttggggggggggggagcgggaggagggtaggggatgtctgtgtgactgtaagtcacatgtgcgcaggtctccatgggagccagaaaacggcatggcatctttggcactagagtgctagatggttctgtgcaccatgtgggtacagggaacccaactcagggcccctggaaaagcaacaactgctcgtgtaactacttagtcttttccagtcccctacattatagtcttaaaaatgttttaaaattaatttttattttatgtgtatgagtgctttgcctgtacttatgactgttcaccaggtacatacctggtgcccacagaggtcagaatggatcatcagatcccctggatctggagtttcagagcaatgagccaacatgtgggtactgggactgaacctgggtcctcttgcaggagcaacaaacgctcttatgcacttagccatctcttcaggcccccaaactataatatttaaagtatttttttccaccagtagacacagtttagatggtgtgtcctagttttcagatgcaggaagtcatcactggctagggaagctgagttgatattttgaaactcatgattcatatgtctctttgtctcactttgcccagctaatgactcccccagaggcaaagaagtatttcaacttcagatatccacctgctggagcagaaagagtattttatggcagagcaaatgacccccaaattgcaccttatttgacacatggactaagatctaaaatctcaataccggtaaacctttctcttttttttcctcctacattaatcattcatttacgaagttagaaagttgtacaaatcaaggtgtaagtgaaattcataattactatgggtattttccttcctcattctggcttctaaaaacactagtcttgggcaatgggttgtgttttcaagtaactaaggataggcgaaggaagcagcagttcaaacccagagtttactgatcctcagatgggatgaggctgcgacattcccattttcccagtaaagaaaaatgttggaatatcttattagccattaagcgaataggcctgctccttttgcccaaggccctacagagagcatatatagatcttgagtggatcagaaaatatttgatgctgactgctgggatataactgtcctctaattgcttatttatctaccttcctacctttccttctatatttgttatctccttttatttatttcagtttattaagtttatttatttagttatttactggtggggctaaagctggaactcggggtaatctgtattctgggtaaacactactgttgagacctatcatcaaccctctatctttttgtttaaaaaccttttcccacattttgtgcgtgtgtgtgtgtgcgcgcgcgtgtgtacatgtgctacagcacctgtgtgggggacagatgagaacttgggacagtcagttctttccttctatcttgtgatttctggagactaaactcagatcatcagccttggcagcaagctcccttacctgctgagccatcttggccattaaaaaaaattcattgattaatttttagcgtgtgtgtgtgtgcacatgcacgcatgtgtgcacgcacgtatgtgttttatgtgtgggcatgcatgtagggacttagaggacaactttgtagagttgattccctctttctttacatggtttctggggatcaaagttacactggcaggctcttgtggcagtatttttacttgctgatccatgcctcagggccttttctgtttttgagacaatgtactatgcccagcatctatctctcttgtctttctcttcctgtctatctctcccctgctttttgtttgtttgtttgtttgtttgaggcagtgatttttctacatagccatagctatcctggaacttgctttgtagacaggactggccttgaactcagatccacctgcctcttcctccacagttctgggattaaaggcatgtgccacaccatgcccagtgctttctctcatttttatcttttgttatttgaagtttttaactctgtagtccaggttggcctcaatcttcctgcctctgttacttaaggactgggattacagtactgtgtcattaggtctaatctgggttttatatgtttgagaaagttggtggaaatgattttatgttctgtgcttgtcatacgtgttcttgagtatttgcatcaataacctggaaactccttccataagtcgttagcaccactgtaggaaagttaaatttatctgtctgtgtatgtatgtatgtatgtatgtatgtacatatgtacatacttaactatctatttacttaatgctggggattgaacccgtggtcttgtacctgttaggcaagtgctctattgctgagctacttctctagtccttttttattttctgctttgtggttagaacttacagcctcatgagcactccactactgaaccattctccatctggtcttgcaatctttctgtagtttcatgcctgcctggagcttagtatcctggtacttcagttcccccaagagctgggatacagtcctagctgaaagttctgcatctgctttgttaaacttttcagtactgatttcattgtcagttcattggaagatgagcacagccaatgatgatgggggaaatagcactttaatcgtattttcttctcattagagttttagttttcttacaaatactggtttcgatacatgaacattctataatttctttagatgggatcattgataaacccacagcccattaccacgtttcaacagaaaattaaagacaaaaaagaatctatatattttagccatcaacgagcaccattgggaaaatctcatgatcaaacaccaggattgccaaaaggaatggatgtgatcaacacaacattagggacaccgactatcagaggtagtaaaaataagggacaggaggaggtgggaaatacgtcttatgaaactggattaaatatgtcactgatatattttctgttgatgatgtgaaggattgttagtgatgaaagatgaacatagtcttcacacaaggtcagtggtctgcttgggccattctctccttctatcttgtcctgtTGACAATGACATCACATCCTCTCTGTCCCCTAACTATAATCTCACGTTTGCATTTTTTGCTTTCAGTTTTCTGCTTTTATTTAAATCTATACTTTTTTTTCTACTTAGTGGGTATCTTGCCATGGTACCTGAAGAGTGTCGCACAGAGAGAGATGACTGAAATACTCCAGGTACAAGTAAAAAGATGGTATCATAGCAGGATCTCTCGCT

This mutation is a 3470 bp deletion beginning at Chromosome 17 position 53,449,206 bp and ending after 53,452,675 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48285 TGG CAA GAT ACC CAC TAA GTA GA Mutant Reverse A
48286 Fluorophore-1 ACC CTG AAA TGA TCA ACG CTT Quencher-1 WT Probe
49191 Fluorophore-2 ACA TCA CAT CCT CTC TGT CCC CTA Quencher-2 MUT Probe
49281 CGT TTT AGG TAC AGC CCA AG Common A
49282 ACT GGG ACG TGC TCA CCC T Wild type Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48285 0.40 uM
49281 0.40 uM
49282 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.