Protocol 36353: Probe Assay - Spatc1<em1(IMPC)J> Alt 1
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

>chr15:76292776+76292870 95bp AGGAACAGGGAAGGGATGAT CTCTACTGTCCTGCCCCAAG

Mutant=  102 bp

Wild Type = 95 bp

Sequence

Partial Wt Sequence (deletions in lower case:
GCCCGGCTCACTCCTCCCAGCCAAAAGTAGAGAGCCAGGTTAGCTGTCTTTGAGTACGACCACTAGGAGccgctcttctggtgtggggttggcggcagagagggacagaccaacatccagaaccctctaaagtggcacagaatgctctctgtgctgaatgcctctggctactccacagatgccctcatccaacccacctaaggaatgatgctcagagagggcaggtcaagccagccagctcagcagacagataccttgaccagacatgagtgggtctttgtgttctttctgtatccacaggtgtattcctgcccccagcttcagcagcagcaaaggagccctgctcagaagacttggggatggtagcactggcacctctggctgacatgttgaataccccacagctcagtcctgcagcaggctcccttgtgaaccccctggctgctactcttaacccactgctgtctggccaaatacccttgttgcagaacaaccagtttgccaatcttgtgccatgctccatgagcaaccaactgaccaatcctactacagtttccccaggagtcaccttggccagttctctgggtttgccctctactggacccctgaacagtcaaatgactagtcccatgactgtacccccagggaccactctggccagctcactgggcctgacctcaactggttccctaacaacaagcagccggctggtggg.........................ttacttggtaatgaaaacagaagttctgggagcggaggaaaaaaaagagggagtaggatcaggaacagggaagggatgatgcagaaagaaaggatacttccctgttcttagCAAAGGGGTAGGGAGCCTCAGGGCCTTGGGGCAGGACAGTAGA.

 

This is a 9510 bp deletion beginning at Chromosome 15 position 76,283,317 bp and ending after 76,292,826 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46672 Fluorophore-1 CGA CCA CTA GGA GCA AAG GG Quencher-1 MUT Probe
48499 TCC TCC CAG CCA AAA GTA GA Mutant Forward A
48500 CTC TAC TGT CCT GCC CCA AG Common A
48501 AGG AAC AGG GAA GGG ATG AT Wild type Forward A
48502 Fluorophore-2 AGA AAG GAT ACT TCC CTG TTC TTA GC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48499 0.40 uM
48500 0.40 uM
48501 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.