Protocol 35709: Probe Assay - Spty2d1<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

>chr7:46997246-46997347 102bp AGTGGAAAAGAGGCTTATCTAGGAC ACCAGCTGCGTCCAGTCA

Mut= 104 bp

Wt= 102 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GAGACTTAGCTCTGTTTGTCATGCTTCTTTAATAGGTATTTAAGAGATTGAAGTCTTTCTTTCAGGGTTCAAATCATCCTTATTCGTGTTTCAGTCACTTTCTTTACGCAGTTACTTAAAAACCAAAGTCCACACAAATAGCATCTGTGCCCGGGCAGGATTCATGACCTTGGGATCTCGGAGAGCAGtgtaaaggagcttgtgtagcaaattaatagttaatgaacttatatgccgcagttgaagagtccagagagctgtgctctttccttagattccttcccttcctcttccttcctttcctcgctttgttttctgtgttttctgcagtactggctggggattgaactcctagctgataaatgtcagacaagcactcttcctctgagcctcacatcccagcccctagactatatatttgtaaagtgttgctgcttatagcttttattctgcttttgtttttagaaaaggtacagtttggcggtggggcctccaaagaaagacccaaaagtgaaaggtgtccagtcggcagctgtgcaagctttccttaggcggaaagaagaggagcttagacaaaaaggtatgcgcttgtactgcatctcagcctgtctgccaccaaatccgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtaaacatcctgacagcttagtagtttggggatgttgtatgtgtgtctgggttaactttatagacaaatatgtgcgtgcatgtttgtaaatgtgctcataggttggttatttgggttgttttttctaaccccatctcacctacccccaaggctttgtggagctggccacagtgatattcacctgtcatcccaggtattcaacaggctgagacaagatcatttattgagctcaggagcttcagagtagctgggacaacgcatcaaggccttatctcttgaaggaaggtggggagacatgaaaaggcctggtgtatcaaccatttaactagccataatatgtctccaattctttaccttggtcctggcattgttatagatattcattgattcactcttaacagctccagtcagtgctggtgtcctattgacatggtatatttatgagccattttgtgcattttaagcaaaagccaagtcttttatctgcttagtcataaagagaagctcatatctgacccacttgtgtagtaattactcagttttgttagatgacttcctcatttgacacagctctctagaaaccataactcagattgagctttgactgtggagtggcagcacatgagcgctgctatgctgacactgttgaggtgatgtgctcttgttttattcctttcagccttggaagagaaaaaaaggaaagaggaactagtgaaaaagcgaattgagctcaaacatgacaaaaaagcaagagccatggccaagaggacaaaggacaattttcatggttacgatgggattcctgttgaggaaaagacaaagaagaagcagctggtggagagccacctgaaccagggaactgaccaggagtatgatgtggaggaggaagacttcatagactacaatcaggctgagctggaccaggactatgaggaggagcaagagccacccaaggctgaaagcaaaccaaaggccccccttaaaagtgccccgtcacccatgaacttcactgacttactgaggctagctgagaagaagcagtttgaaccagtggagatcaaggtagtgaagaaagcagaagaccggcccctgactgcagaggaactgagggagcgagagtttcttgaacgaaagcataggaaaaagaaacctgaacccgatgccaaactacctccacccgtgttgaaaaaggcgccctctcataaagacatcatgggtacaaagcccagcagaggtgctggggacaggcagcttgcttccaaaggattgccctttcctcaagctgagaagaaattcagacccagcacggccagtgagaaacaagcagctttgtcttcacccaaatccctgccaggcgagaggaccaaggtagggtctggcagtagcacccagccctcacttcgggagggtcacaacaggcctgtcttcaatggggctggaaagccccgccccagcacctgttcaccaagtgtcccaaagactcctgctagcgggactcagaaatctgcttctgagcacaaagccaaaaaacctcttccttctcatccgagccattccaagcctggacccacggttctctcacacaacaaatctaagagtccaggtgtcagacaaccaggcagcaactctggctctgctcctgggcagcctaacccagggacagctcgacccacacttagttctggccctgtgcccaggcgccagaacggcagctccagctcaggacctgagcagtcagccggtgggatcagaaagctggccagcaattcacatctctccggacgaacactcaacggcacaaatggtcctggacgacctgcaagcagctcaagtggccctgggcgacccatcagtggatctgctggttctgggagacctgtgggcagttctggaggccccgggcagcctgtaaacaatccacatgaccttcgacgaccaatgaacagtcttagctctcctgggcgggcagtcagtggccctgggagatccataagtggttccattccagctggacgaactgtcaattcaggccctggaagaccagtgagcagcctaggccctggacgagcagttagtaacccaggcctccccacaaagcccagatgtactgttgtctcagaaacaatttcttcaaagaatattattagtcggtcaagtaatggacagataaatggaatgaaacctctcctctctggctacagatctgctcaaggtaaagttgacccttatcccaaagagtatatttatcactatgcctttggagaaagtacctagctttatataatcaggaaccctatgaccactttcacaagccctgtaatgtgctcattgttgatagtggaaaagaggcttatctaggacccgctgttcacaccccccacctctttctcacatgcagtattgtgatgtgtgagAGGGGGTGACTGGACGCAGCTGGTGGTTTGTAGGATAATGTTCTACATGAAGACTAAGCCAGATGAGAGGATGATTAAGAACTTAGAGCCTTTGCTGCTCTGTCATTCAGCAGGTGTTTATTGAACACCTCCCCTGTAGCAGCTGCTATTTTAGACTGTCCTGAGTTCTATTCCTTTTCATGGTGGTG

This mutation is a 2893 bp deletion beginning at Chromosome 7 position 46,997,270 bp and ending after 47,000,162 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46823 AGT GGA AAA GAG GCT TAT CTA GGA C Wild type Forward A
46824 ACC AGC TGC GTC CAG TCA Common A
46825 CAG TTA CTT AAA AAC CAA AGT CC Mutant Forward A
46826 Fluorophore-1 CCC CAC CTC TTT CTC ACA TG Quencher-1 WT Probe
46827 Fluorophore-2 AGG ATT CAT GAC CTT GGG ATC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46823 0.40 uM
46824 0.40 uM
46825 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.