Protocol 36352: Probe Assay - Smim20<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

>chr5:53276821+53276927 107bp TTGTGCCTGTCCCTTCTTTG TCTCAGCATTTTGGAAAGGAC

Mut= 103 bp

Wt= 107 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (TG insertion):

 ATGCTTGATTTCTCTTGGCCTCTGCCTGTTGTGCATCTCCAAAGTGCCAGGCTTAGCTGCTGCAGCTACAGCAAGCTCACTAATTGGCACAGCACCTGCAATCAATTAGGGAAGCTTCAATTATCCAAACCAAAGCCACCCACCCAAAGATTAATTTTCTGCTCTATTTCCTATCAAAAACATAGCATATTTAGCTTGGAGTCCAGTCCTTGAGTGGAGTGTTTTTAGCCACCGTTTTTCTGATAATTGTGCCTGTCCCTTCTTTGCGGCAGCTCTCCTCT^tgtaatgtcacacccaccagctctttgaaacagttctttctgttgtgttgtgtcctttccaaaatgctgagatcacaggaaaaagatgttggttgggatctttttgtttgaagtgtggctctttttacatctaggatctagcttctataacctaaaagctttaagtaatgacgtccctgaattgaatcacactcctaaatatgcttgtaattagcccatataaatagaagcaaaatgcccacatctcctttctaaagagtaattatttgttctgttgtgtttctcagagaaggagcaggctgtaaatcgagctggtattgtccaggaagatgtgcaaccgccaggtaaactgaaaacgctcagacaatcccttccggggcacgtggatgagtactttgctgtggagtgggtggttcttctccctggaacctgatactgttcattattttatgatagttcctctcttgtcagggctatctaagacttcatgctaatatttcagtgcctagtactttttacaagcttcctgggcccctcgtttgtccttggtaagtggacataaaatggaatcaagtatgcaacagatcatctgctttgcagactcccatactgagcacccctgtaaatacctccctcactggcagaccgtgtgtgagggtgagcactaaggttcagtttcctagagaagttctcaaagctcccttcagggacgtgtgggatgttcctttaaaatgatacaagttggctgagtacgagagattttctagcttattaaccaaactcctgcagtctggtggtcctaggctagatccatttcttcctagcagggaagagttcctaagcctttaaaaggatacctcatagctgccatggctgcctcgttgcagggttccagtggctgcactcacccatttcaatgtctcagccacatggaaattgcatggctcaatttccacatcatcttgtttgtttgggatcaagggggtgtgttcagtcatttagcaatgaatcctgatagggtatttgtgtttctttccatagggttgaaagtgtggtctgatccatttggcaggaaatgaggctgtcagcaagtctgatgaggaaagtggacgtctttatcctgtgcactccgcagtggggacaatagatgcctcactgtggcagcatggcatggagagggaactctcatgctgctagccagaccccttgtgatagagactgtgtgcaaagacagtgcttcccttaactccctggagaacctgaacagatgccaccattaggaagtgccttgcggctccattgactttgcaggagcagagccagcctgcaaggctgtttgtggaagatctgctgctcctgcagtctttatcacttccaagctgtgatgtgaacacaagcaacctgtgggctcaaggtccgtggctgctctgacaccttctgaataagcgatttcagtgcaaatggccttgccaagctgcctcgcagggctcttggaggatgtttcagttgataaaactgtttgaagacaggatccttggcactgtttaagaatatacactgctcagcttaaccatctcattgaaagtcactgtgtgtggaagtgaatagggagcgagtcacactagactataccacacacagtagactcctgcgtgaggctgcaggtattaaaatggtttctcttagtttgctggtgtgtactgtttttatattttgtgtgtgtatatgtttgttttatttggatgggtggtgtttgttggctttggattttgttttttgagacagggtctgtcagaccagtcaagccttgaactcccagagatccacctgtcactgcccagtagaatttttttgtaaattgaattctgatgtgaagaagtagaagtagtgctattgtctagattctatttgagtagatatccgtgtagt^AATAGCTATTTGTAGACAAGGTGAGCGGCACACTTCTTGTGGCAGCTGTGGCATTAGGTTCTCCAGAGAAACAGAACTGATGAGATGAATATGATTTACATCAGTGACTTACAGGCTGTGGTTCACCTAATCCAACCATGCCTGTCTTATCAATGGAAAGTCCAAGAATCCAATAGTTGTTCAGTCCAAAAGGCTGGACTTTAGTATCTACCAGAATCCCGAAAAAGTCGTCTGTAATCCCCGTGAGAGCAACGGCAAGCAGGCAGAGAGCACACA

This mutation is a 1929 bp deletion beginning at Chromosome 5 position 53,276,856 bp and ending after 53,278,784 bp (GRCm38/mm10). In addition, there is a 2 bp TG insertion at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48494 TTG TGC CTG TCC CTT CTT TG Common A
48495 TCT CAG CAT TTT GGA AAG GAC Wild type Reverse A
48496 CTG GAG AAC CTA ATG CCA CAG Mutant Reverse A
48497 Fluorophore-1 AAT GTC ACA CCC ACC AGC TC Quencher-1 WT Probe
48498 Fluorophore-2 ACA AGG TGA GCG GCA CAC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48494 0.40 uM
48495 0.40 uM
48496 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.