Protocol 36789: Probe Assay - Aftph<em1(IMPC)J>Alternate 1
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

>chr11:20727690-20727833 144bp TGGTTTGGTTTCTTGTTTGG CATAACAAGGCTTTTCAAGTTTC

Mut = 157 bp

Wt = 144 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 CACACTAACACAGGAACCTGAGTTTTATCCACTGGAACCTTAGGAGTGAATAGGGAATAGGTGGTTCCCTAAAGCCTGCACATTCCAGACCAGTAGGACATCAGGTCTCAAAAATCAAAAAGGTGGACGGAGCTCGAGAGACAGTATGTGAATTGCACTCGGAGGTACATATGTACCTGTATACACACATGGAAATACATATACACAGAGTAGAGCAATGTAATTTTCATTGTTTTGGCGGTGGTGGTTTGGTTTCTTGTTTGGTTTTTGAGACTAATGcccaggttgactggctcaggatgaccttgatgctcctcctggcactccctgagtgaccttgccagataactttgtattgttttaaagaaacttgaaaagccttgttatgtaatctgcctttgaaaataaacaaaactaatgcttttcctttttttaaaggcataaactaactactggaaagctgcggaataatggagccagacatcattcgaatgtactcttcctcccccccacctctagacaacggagcagaggatgatgaggaggatgagtttggggagtttggtgggttttcggaagtcagcccttctggtgtagggtttgttgattttgacacaccagattatactcgtcccaaggaagactttgtaccttcaaaccactttatgccaattcatgaatattcagaagatgtagatagccttacaagctttaagtctgttcagaatggtaatgataaggatatcactgctgaactttctactcctgtgaaaagtcaatctgatgttgtactttctaccaccagcaaagaaatgattccatctaaaactttggatccttccatcgatggtatggaaagcctggaagacttagataaagtagtggtgcagggaccaagcactgggcaactcagaagtttctctcctggagattttagaactgataagaacattgttcaccaaaccaagcagttagagagctgcaacggtgaaaagcctccttgtctggaaattctaacaaacgggtttgcaggtttggaaactgtaaatcctcagggaacagatgacctggacaatgtggcagattcaaagggatcgaagcctcttaacacttgtggcaccgagtgcattttagagtctgcagctagtcatgctacggaatttgcagatttttctacattttcccaaacagaaaggacacaactggaagaaatagaatgtccagttttaaatgatggtgatacactgaccattcagggaaacagcaaaggccccagagtcaaggaactaaactgtgtgaaagaagtgactttggatggaagctttgaggatacggggaacactgaaagagaacatcaggtctgcgtctcagaaatccatgccgtagctgacagagggctcagtgttgaaaaacaagaccttcaaacactgcaacaggatgagtttctaaattcaaggattcagtctgaggcttggagcttggtagactcaagtgaaaattcagaagccattacaaaggagcggtgtaaaatggaaaaaaatgacttatttgcctctaagtgtgctgacttatccatggattctgttaaaacttcagacgttaatgaaattggttcttccaaagaagaaaacagaaagctgactaatcccaaaagcccagaccctgatcccacaggacaaaatgctttggatgattctgctgcaagcatgaaaaatggtgacagtgggaatggctttgtgacttgtcatgataccaatgaggatgattttggtgactttggtacagccaatggcaccactccaccctttgttactagtactcaagattcaatgagtgacgtcacttttgaagactcctcagagcattttctacatcttagtgaaccaggtgatgattttggagaatttgaggatacaaatgctgtttcttgtcaagaggaaatgagatttactgagtcagacctaagacagacttctgatggcttatcagaggaatgtccattggcaggagagtctggtggaaaagacagtaaacctgactcaaaactgaaaaatggtcaagacagtgaatttggagattttgattctgtgccaaatactcaaggtagtgcttttcaagactcggatgattttgcagacttcagttcagcaggtcctagccaagctgtagattggaatgcttttgaggatgaacagaaagatggttgttcttgggctgcctttggggatcagcaggagactgagtctcatcatctaaaggaagtctggcagtcacagaggacagatgaaactatgggcactctgggaacccctaagatgcacagtgtttcttcagcagcttccaaaggagcagttgctagtggccatttgcaggaaccaggcacttcagttcaggtatgtacttactgcctctgatttgtagaacatattaattgccaaggagacttctaatacagctttaaacaaaattacctgagtatctgtcaaagaggtttctgtgtgatatatgaggtttgttggtctggtttgcacattagttagatttttcacaaccctttgaaaaattgtaaaactgctacacagggagtgtcttaactagttaacaggaatttatcaatttaaattatccatggaTAGTATCTGTTTTAAATAGTAGCCATTTTTCCTTCTGTTTTTATCTTTAATAATTATTTCAATTAACAGACTGGTGAAATACTTTCAGTTTAGAATGGGTTTCTGAGCAAAAGACAACTGGGAAAACCCTTTTTTTGTTTGTTTGTTTGTTTTTTGTTTTTTTGAGACAGGGTTTCTCTGTGTAGCCCTGGCCATCCTGGAACTCACTCTGTAGACCAGGCTGGCCTCAAACTCAGAAATCTGCCTGTCTCTGCCTCCCGA

This mutation is a 2354 bp deletion beginning at Chromosome 11 position 20,725,445 bp and ending after 20,727,798 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48889 TGG TTT GGT TTC TTG TTT GG Common A
48890 CAT AAC AAG GCT TTT CAA GTT TC Wild type Reverse A
48891 CCC AGT TGT CTT TTG CTC AG Mutant Reverse A
48892 Fluorophore-1 CAG GAT GAC CTT GAT GCT CC Quencher-1
49709 Fluorophore-2 TGG TGA AAT ACT TTC AGT TTA GAA TGG G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48889 0.40 uM
48890 0.40 uM
48891 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.