Protocol 42145: Probe Assay - Blcap<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

Mutant=  120 bp

Wild Type = 114 bp

chr2:157556069-157556182 114bp CAGGCATTAACAGGAGGGAGT GGGATCCACTCCTCAAGTCC

The  Wt For primer (primer 59969) anneals over the nucleotide sequence containing mouse genomic variations rs27338091.

Sequence

Wt Sequence (deletions in lower case): GGCTGCAGCCCCAGGAGGCGATACATGATCTCTGCTGGGCAGGGCCCAGGCAAAGCCAGCCCACTGTGGGGACCGTGTCCTCCTGGGATCCCCCAGTAGAAGCctagggaagacacaccctagcaggctatctttaagtaataatttggataggagtagacattcatgggtgggtggggaggaaagggcattcacatttgctgttgttaaccatgatccattttcccaaataagtatacaatttgttcctgtttttttgtttgttttgtttgttgttttttgttttttgtgtgtgtctgtctgtcctttgtctgtttgttgtgttgacttttctcttcaggatccctgctgccttggtgatcccgggctgacagccagagagcacagcggctcagctcctggagagagagtcgaagaaagcgaagggcagccacctgtgcctgctggctcccattaggtcggttcctgcagcggtgcctggcagccttggtgaaggccctgcccggcagagatcatgtattgcctccagtggctgctgcccgtcctcctcatccccaagcccctcaaccccgctctgtggttcagccactccatgttcatgggcttctacctgctcagcttcctcctggaacggaaaccttgcacaatatgtgccttggttttcctggcagccctctttctcatctgctatagctgctggggaaattgtttcctgtaccactgctccgattccccacttccggaatcagcccatgaccccggtgttgtgggcacctaacgtctgccgagatagcttgccaaggaagcagaagacgggaggggaggcattgacataggtcataaagcattggagtttcaaatcccgcagtcctgcgggtaccacattcctaatggagccttttggcctgtgatgttttatccttacaatgtgaataatggcactgatcggtgcttctgttgtagagtcctgtagtcgtgggtggtcttatggttgtgtgttctgtcaccatctgggtcccggctgacggattggcccacccccttgctcattgatttggggaatctatacccttgatatgacctgtgtggatacagtgtagtctcaatgtcacctccataacccttcctcgtcaagaccttcctcgttccctcctcgtcccctttccccgttttccccttggttcacttccaacccctttccttttttggggagcacctgtccaagacagggcttgtttttgcacttatctcaaatttgaagagattgctgatgcccgagagcctcgctttttcatccttcgttcccttttgagaaggtgagacggaatcatgtctcaactgctcgttgtctgcagacctccagtatttcctctgcctcatttttaagaaagaagcgatggggagacattgctctttggcctgggtatctgggctcctgccttccagcccagcctctctccctttgctcttcctcctgtctctctcagctgacctaaaggggccacctcatgtctccagtgcatgctcttcaggagggagatgtgcagtattctcgtagacccagtggtccctggctgagtgaatgagaaagtattacatttttcatagcagccatgattcccttggtaggtgtttggatatttttgatgtgccctgtatgtatgtgtgcactagtgtcagcgtgtatgtacacacgtatgtgtgaatgtgtgttgtgtgtgtgtttacataccaatacatgtgtatattccttttgaagaagctttattgaatatgttctgattttgaggtttagtagtagtagctagctgtagtaggtcctgctgcagtttttatttagcatggggattgcagagcgaccagcacagtggactccaaggtggttcagacaagacccaggggagcagtcgccatcatcctcccaccaggagcttcctcattgctgcgcacgtagactgtacactatgaagaaaacacaggaagaaagatttggtgacttggtacttgtttgcttttctctgcgcttcagaaacaagtgtttgcaaatgagactttctcctggcccctacccactggggatcagcatggttgttcttccagtcggaaatgtacccccctcctttccccctcttgtgtgcaagtggggggggggggcaggcataggacagagctggaagcaggcttctggggagtgggacttagaggccacacttgtgaaacactcggactgctgttgtaaagcttttatttctggtgtgttcgttccacagctgtttgaaatgtttaataaagctttataaactttactttgtggttttatgtggctgcagtccatttgggtcatggtgttggatttctgagttggtggggggcagggtgaagcacacagggctgagtccttggcgccagagtaccttagctggaaaatctgatctgttctctggagaacagatgctggaattggggaggaaagcagggggcagctcaggcattaacaggagggagtctggaagttttcagagatgccatttgcacttctcagctcccatggcttcctgcctgaagcTGTCTGCTCTATAGGACTTGAGGAGTGGATCCCTCGATAAACAACTTGGGACAGCTGGGG

 

This is a 2463 bp deletion beginning at Chromosome 2 position 157,398,022 bp and ending after 157,400,484 bp (GRCm39/mm39).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
59969 CAG GCA TTA ACA GGA GGG AGT Wild type Forward A
59970 GGG ATC CAC TCC TCA AGT CC Common A
59971 GGC GAT ACA TGA TCT CTG CTG Mutant Forward A
59972 Fluorophore-1 ATC CCC CAG TAG AAG CTG TCT G Quencher-1 MUT Probe
59973 Fluorophore-2 AGC TCC CAT GGC TTC CTG Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
59969 0.40 uM
59970 0.40 uM
59971 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.