Protocol 41646: Probe Assay - Fbrs<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

>chr7:127083522+127083666 145bp AGAAGGGGTTGTTGGGTGTG TCTAACAGGAGGTGGGTCAGA

Mut= 157 bp

Wt= 145 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

GTAGTAGGATAGGATATTGTGGTATTGCTTCTATTATCTTAGGGTGGTGGCTCTGGTGCTCATTGTAATTAAGCATCAAAATGAATGCTCTGTTGTATGCTGTGGTTGAGTCAGAAAAGAAAGTCATATGGCAAGTGATGACAGAGGCATCTTTGTAGAAACTAGATCTAAAGAGGAGAAGGGGTTGTTGGGTGTGGGAGGCATCTCATCCcgagaccggattctctgcagggagtccctgggggccaagttctaccatctcccatgttggagaaccaaggccacatcaccacccaggcttctgacccacctcctgttagagctagcacctcccaccccctcactcatctccccacttctctccccagctcttcacggccgccccccaaggccccggcccctcccgtggctcagcctcccccctcatcatcctcttcgtcctcctcctcctcatctgcctcctcctcgtccgcgcagctcacccaccggcccccgacgccctcactgcccctgcctctgtccacccacagctttcctccccatgggttgcgcccaccaccaccccaccacccttccttggtctcccctggccccaccctgcccccacccccacctctgctgcaggtgccagggcaccctggggcctcagctgctaacgccctttctggtgagtttggggtcctggcagggtggagtggtggggggccatggccccaaggatgggctcagttggctttggcaggaaccaagggcctgtggagggagtggctcaaggctaaagggaaggccagtcaccttggcccaaagaggctgactcggtggcgccagatattaaagccttcttcccctcccctcccctcccacagagcaggacctgatcggccaggacctgaactcacgctacctgaatgcccagggtggccctgaggtggtgggagcagggggctcggcccggcccctggccttccagttccaccagcacaaccaccagcaccagcacacccaccagcacacccaccagcacttcactccctaccccccgggcctgctgcctccccacggccacatggtgagctcctccttgggctgctgctgaggcctaagggaaagtgtctcctacagggaaaggaagaccctggccccctggctgccagcttactcttcccttttcttccctagtttgagaaatacccaggaaagatggaaggccttttccgacataatgtgagtgtgtgtgagggtgtgtgaagtgtgggaatgtatgtatcagtggagtccagtcttcacctccaaaagcacgaaccctgagcttgagaaagctccttgggggtgttagggtggaagccaaagaactaatgggtagagcagacctgggctgcatctccactcccttcttggactagtgcccttctctccacagccgtacatggccttccctcccgctgtgcccgggctccctccgggtctcccaccagctgtctcctttggctccctgcagggggccttccagcccaaggtgagctcctaatccagacaactactacctcccatcccttataaacatcctggaaccctttacctgtttcccattccccaaaatgtttctagcccccattctcccttctggacccaagtttttgcaaagccacgatccctctcttgaccctttttccagcttagttctagacccctttttaacttggtactggctctgctgtctgatccctacactccccttcactcccctttcccagaacacgaaccctgagctgccaccacgactggggccagtgctgagcgggctaccccagaaggggacacaggtgaggggggcagggcaggtcctgggggagttggaaagtatgctgagggaaagcagaattgacgagagagggtgagttcttggttcttcccttaggtgtctaaagagagaacttttgtctaacaggtctaccccggCATGGGAGTAGAAGGTTGATGATATTAATAAGAAGAATGAAGTAATTAATAGAGCAGTAGTTTTGTATTTTAGTTTTCTTTGTTTGGTGTGATTCTAGATATCAAACCTAAGGCCTCACACATGCATCTACTACTGAGTTATAGCTCTAGCAAGACTGATTTTTTGA

This is a 1748 bp deletion beginning at Chromosome 7 position 127,083,557 bp and ending after 127,085,304 bp (GRCm39/mm39). 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
58833 AGA AGG GGT TGT TGG GTG TG Common A
58834 TCT AAC AGG AGG TGG GTC AGA Wild type Reverse A
58835 TGT GTG AGG CCT TAG GTT TG Mutant Reverse A
58836 Fluorophore-1 CGG ATT CTC TGC AGG GAG TCC Quencher-1 WT Probe
58837 Fluorophore-2 AGG CAT CTC ATC CCA TGG GAG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
58833 0.40 uM
58834 0.40 uM
58835 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.