Protocol 34000: Probe Assay - Apobr<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

>chr7:126585346+126585475 130bp GTGCCTTCACTCTTCTTTCTTC CCTCAGTCACCTCTCCCAGT

Mut= 136 bp

Wt= 130 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GTACCCACTTATTCAAGCTAGGCCTTGTCGGGGTCCCTTAGCAGACGTCCAGATCCCTGATTCTTACCTGGGCTTTGTTAACAGTGTACTCCTGGGGAAGTGGTTAGACCTCCCCTGAGTGCCTTCACTCTTCTTTCTTCTCTTCTAGGACTCcttcagtgcgtttgtgtcctacctcgtaggagacacagtccccacagtagagaggcagacacaggcagctgaggaactgggagaggtgactgaggggaaggttgtaggagaggaagcccaggaggtactggagggacttagaagtggccagagtgagggggtagaagcacccgaagagactagaagatgccaagaaggaagtttagctggtgaacagacctgggggtggagagcagatagctctgcaaggccccaagcagaaaggcaggacactgggtcctggaaggcagctgaggatgccaggggccaggagccaagtgtcccactgaagcctgaggcagagcctgggactcacagagacaggagcagtaatacagcccaggagatctgggagcacggtgaagaggaagcaagcagtggggagcctctgagaacctgtgaacagaaggaagaagaggaggaggtggtcagagcagcagagtcagggatggccgagggggtggagtcacagccaacctggcacagcgagcctggggggaatgctggcactgaggggcagcatgtgacagaggacagcaaagagatagactgggtggccaaagacatggttgcagaaatcgaatggtttggtgccaaagggattgacaaggaagaggagaggatggtcccaatgagggatggtgaaagggcaagggcacaggggacacaatgccccggggccgagtctgaggaccaggccatgttgagtagagaggcctggacagtctcagacagggagggggctgatagcctaggagttcagggaacagaatacgggtcagacccaggagacaacttcccaggaactactgggagagtctgggtcctagaagaggctgacaagggagaccagcaggatgaggtcgacgagaagagagaggctgaagttaggtttccgatccagaccctggaggctgagagaactggagagatgactgaaggccacatagctgaagaggaggctatgggagaacaggaaacagagggcagctttgaggatgaggagaggcaagacttagccatcagggataatggagtgagtctagaagaagaggtacgggcagaagaatcctccagggagaaaaggaactcctgggccacagagcctacgctagtcctagacacggaggctaaagatgaacctgactgggaagactctccagaagtcagcactgaggagttgtttgtgggagagaggagtgaagccgcccagatgacaccagaggtattgagagtaaaggtcactgaaggacaggaccctgagctggtgagacattcccaggctttaactaaacaacttgaggaaggacagaagggtcaggaagagactagtggagctccagacctgagcccagagcgggtgctaagcttgaaggaatatcctgggcctgtggggtttgcaggccctgagctagaagcctggggaaactggagtaggggtgtggaccgcagaaatagccaggaggtaaaagcagatgctgaagcaggcaaggaacagactgcaaccgagcaggcagtggagatccgggctgaaggaggccaggaggcccaacagccagaggtcttcgggtcaggaggagaggaggcactgacctccgtagcacttaacccggagctggagggaagccaaggagcagaggcggggacagaagagtcagtggaggaatccaagcccacagaaaacgaggctgctgaggaggaggctgtagtgccttgggaggcagatggaacttgcaggaagaggaggctggaggaggtgactctgagcctgcaagacagtgaggacacagagaccagttatttggctgaggagattattgtgggtatcagggccgtggataccgaggaagggccaaaatgggaagctgggttggctccagaaacagagcttgggaaagcctggtgttccgagggcagaggggaggctgggagaggcacggagctggaggagactacagagaagcaaagtggacaggaagttggcttggtgggctcagcagagaaggtgtctggctatgatatccaagaaattgatgggactgaagagggggagcaggcagagatggagacatctgtaatggcagaagacataagggggacggatggtgtgactttaggctcccaggcagagagagcagaggggtccataacccccatggagactgaggggctcctaagagatcagatgctgttggaagaagaggctgggggagggcagtcgcgagagcagaaggtccacaactcagagggcgagatccaaacgctggacgactcttcagaccaggaggggcagcagacacaccaaatccccacagtagctgtgcctgggcccttggaatcggctgaggccacggcaggtgccccaggggatgttcACAGCAACTGGAATGAGGTGAGGGCTTCAGCAGGGCCCCGAGAGGGACAGGCAGAATCTGGAAGTCAGTTCTCCACAGTCCTCTATGTCCCTTATCCTGCAGGCCCTGCTCCCTGGGTCCCGCCTGGATGTCTCTGTCCCTCGGAGTCGTGTGCTCCTCTC

This mutation is a 2414 bp deletion beginning at Chromosome 7 position 126,585,381 bp and ending after 126,587,794 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42625 GTG CCT TCA CTC TTC TTT CTT C Common A
42626 CCT CAG TCA CCT CTC CCA GT Wild type Reverse A
42627 TGC AGG ATA AGG GAC ATA GAG G Mutant Reverse A
42628 Fluorophore-1 AGA CAC AGG CAG CTG AGG A Quencher-1 WT Probe
42629 Fluorophore-2 CTA GGA CTC ACA GCA ACT GGA A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42625 0.40 uM
42626 0.40 uM
42627 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.