Protocol 34674: Probe Assay - Fam222a<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=  74 bp

Wild Type = 88 bp

Sequence

Wt sequence with deletion in lowercase

GGTTGGAATCTGAGTAGTGCATAACAGCATTAGGGTACATGCCTGAGAGGGACAGAGGAATCAGCTTCAAGGTCATTCCTGTATAAGCATCAAGTTTGAGAGCAACTTGGGACACACACACAGGTCCCTATCTCAGAGAGGAAAAAAAATCGCAAGCTTGCCTGTGGCCGAGGACGGGCGTGCATGTGTGCAGTTTCCTCTCTGAACCTTAGGTTTTGGTCCAGAagttggaagtgtgggaagaacggggaaaattgagggaaagggaccgaccaggcaactcctggaggcggtggggtgtgaggggctgtggctgccccgtctctcacttgtgcctctgtccctgcaggtgagtcagtggccagctctatgcattcctcgcgctaccctagcccagcggagctggatgcctacgcggagaaggtagccaacagtcccctgtccatcaagatcttccccaccaacatccgagtgccgcagcacaagcacctgagccgcacggtcaatggctacgacaccagcggccagcgttacagcccctaccctcagcacaccgccggctaccagggccttctggccatcgtcaaggccgccgtctcctcgagtgctcatgcgcccgccggccaccccaaaagcgtgctcaagagtgtggagggcaagcggactaaactgtcgccggccaccgtgcaggtgggcattgcgccttacccagtgcccagcaccctggggcccttggcctacccgaagccacccgaggcacctgcaccaccgcccagcctgcctgcagcagccactgccacctctgtgatccctctgcccggccggggccttcccctccccccctccaacctaccctctatccatagcatcctgtaccagctcaaccagcaatgccaggccccgggtgctgcaccaagcgcctgtcagggcgtggctgtgccccaccccagtccagctaagcatggcccagtccccagctttcccaacctggcttactcggccacagccggtctgccagactgccggaagggcacagaactgagccagggagccaccccagccctgacactggctggggccaccaaacctgcagggtatgcagaaggtggcctggattacctgctgtggccacagaagccacccccacccccaccccagccgctgcgggcctacagtagcagcaccgtagccggcaagtcccagtccccagagatttgtggcgggagggccttcgaaagggccaacgggtcgccccacaactgcggaatggggctgcccggcagcttcaccgtgggccagtacttcgctgccccttggaacagtgttctggtgacacctaccagtgactgctacaatccagcagctgctgccgtagtgacagagctggcaccaggagccgcccgggagctagccgggcctcctggagacgtactctcgggcctgaccagcaagagtgtgtgtaacacagcggtgctgagcagcagcctccagtcgctggagtatctcatcaacgacatccggccaccctgcatcaaagaacagatgctgggtaagggttacgagacggtggccgtgccccggctgctggaccaccagcacgcccacatccgcctacctgtctacagataaggtctgccctgcggacacacggacagggtgatgggggttgccaggccatagcacagggcgggaagctagcagggcgccccgcccgccctgtgtccacggggaagccaggctggttacctcctcacttcagtgggcaggcgggttgacctcacacaccagggcttctttcctctcccgcttgctgccccaggcagcagggagggtcccaggctggccacgaggctccttcatctttcatccaggcagggaaagaggaccttctgccaacaagaagttccttctgggtctccagcagagggcgctccagtcatcccgtgtcatacacggggtctctatccacaatctcccagtccctgggtccaaagtgccccaccccaccaactaatccaggtgaagaccgccctgactccacacccccagaggtctcaggactaccccccccccgttcctgctgactctccccctacccacccaccctccctactaggaatcacgcccaacctacccggtacgccccagaccaacctcttctccatccctacactacatctcacagggctgctgggaacagttggtgcgttgcacaagggcaccttgcccaaggagactccacacacatcctagatttgtgtatttattacagttgtctgcatttcaaggaagggacactatcttcctattcgcacaaagatgtcacggggggctaacaagggacctgtaaattcaggctccattcacaaggacggtcttgccttcttcgcatctctgggtctcctggggaccagtgggagggtgacctggctgtaactggaatacccaatccagctcccgttctcattccccccacccccaccgcactgctgcggggcgtggggaagagagaagcccaggaagtcctcaaggcctgcagtccccgggccctgccggccccaggcgctccggaaagcggtactgtatctcgccaaggcctgtctgagcactaagtgcatttacaaagctctgagaatgttttttttatactaaaattgaccattatattctactgtgagaagtgcagtctgtctgcactatattgttttaaaaacgaagagaaagaaaaaaaacggaaaacagatggtgtctcagctgtgggtttgctgtaaggctgttctctctctctccctggcttcccccacctcagggatccacgggagggcaccccaaggagccctgtggggacagagtggggtggcatgcagggtgagctatgcccaacttattacaaagctcttcaagataaatgctatcccgtgtctatgtgtcaggcccgtggTCCATCCATAGGGGCAAAATCAATCCTGAGCCCAAGCCCTGGTGGCTCTTTTCGGTGCTGAGGTGGCTCTGAAAACTCCCCATTGGAAGTCCATCTCTAGATCCTGTTGTCATCCTCCCAGATTAAATGGTTGCCATGTAGCTGAGAACAGGCTAGCCATCGCCCTGGCCGGCATGGATTATGCAGGCTGACACCTGGCATATACCCATACCC

This mutation is a 2716 bp deletion beginning at Chromosome 5 position 114,610,703 bp and ending after 114,613,418 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44244 TGC AGT TTC CTC TCT GAA CCT Mutant Forward A
44245 GCT TGG GCT CAG GAT TGA Mutant Reverse A
44246 CCA CCA ACT AAT CCA GGT GA Wild type Forward A
44247 GGG AGA GTC AGC AGG AAC G Wild type Reverse A
44248 Fluorophore-1 CCA GAT CCA TCC ATA GGG GC Quencher-1 MUT Probe
44249 Fluorophore-2 CCG CCC TGA CTC CAC ACC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44244 0.40 uM
44245 0.40 uM
44246 0.40 uM
44247 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.