Protocol 36918: Probe Assay - Msantd2<em1(IMPC)J>Alternate2
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

>chr9:37517492+37517638 147bp GCATGTGTGTGTGGCTACCT AGCATGACTTGGCAATTTGA

 Mut= 147 bp

Wt= 145 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 ATTTTATTGATTTGATTGCTCCTTCAGTTGTAGACATTTTTACCGTGACAGAGTAAGTTAGGAAGGAACAGACTCTGTAAGTGACCTGTTTTGTTAGCTACTTCGTCTAGGCAGCTGTTTTTCCCCTGTACTTAACTAAGAGTAAGGAAGATGTTTTCCACTTGGGGAAATTCTTCTTTCTGTCCTCTTTCCCTCACATTTTCCAAATTCTGGGCACCATAGCAGCTCTCTCTCCTGTGGCAGGTGTACATCCTATTGGCTGGCTGGTATTTCTTTTTTTCCTCCCCCTTATCCTTTTTAAATGGGGGAGGGGGCATAAAATATgtgtattggatacatgcaataatgttgtaatgtttcttgttgttttatataattttataattgtaaattaattgtttgaattataacatgtttgagtaaatgctaaattagtatttttctaatatgataatgcatttgaaatagtattcctgtaacaatgtgtctgtgtttgtctgtctgtgtctgtctgatagtaattaatatctgtggtccgtatctggaggaggaagtacagctttaaaggaataacaaaagactttgtgtcttagacccttcgcaggtgttacagtcgggtgaaagaacatggtgttgggaaaagaaagagcagttacacatttgaacagttggaacaggtgtttggtcagggaggatgggatgctcagccctgccagcctgtacttattaacagtagtggcttgtaccaggagctggagtcagatggcagcactatggaggactattcacaggaggactggggaaaccacagtcaggagctccatggctatccaacagatcaggaattgggtaagaaaacaacatgtgtgcatgtgtgtgtggctacctgatagtgcaatgaatggtatttataccatctgtaatggcttctagtgttgtcagcacaaggcatttgccaaactgtttcagtcaacatggacatccttgatttaacatcaaattgccaagtcatgctcactattagctacgtggtgacttgcatactgttaagttccttgaagatagagacttcctaatctgagtcatacaggagGTTCACTGACACAATCTTATGCAATTCAGAATACCCCTGTCTGTTCTTTGTAGCACTAAGATTTCCTGCTGAGAGCACTGACTCTTGGCCCAACTTCTTTAAATCCACACTGGTCCAATTAAGAGGCTACATGTAGTTTTTGTTTGCTTGTATCCCTGTATGTCAAGTGCTCCAGTTTCTTATTTGGGAAGTATTGATCAGGTTTTAGCTATAGCCTGTGTACATCAGCAGCAAGTTGTGTAATGAGTTTTAGTAGGAAATGCCCTTCCGCCTAGTGAAGTGCTCAGGTGCCGCTGAGAAAGTTCAGGCAGGATTGGCAGTGTAAATTTGCTTCCCTTGCTTAAGGGAAAAAAATACAGAGA

This mutation is a 769 bp deletion beginning at Chromosome 9 position 37,516,948 bp and ending after 37,517,716 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49361 ATC CTA TTG GCT GGC TGG T Mutant Forward A
50035 GCA TGT GTG TGT GGC TAC CT Wild type Forward A
50036 AGC ATG ACT TGG CAA TTT GA Wild type Reverse A
50037 AGC AGG AAA TCT TAG TGC TAC A Mutant Reverse A
50038 Fluorophore-1 AAT GGC TTC TAG TGT TGT CAG CA Quencher-1 WT Probe
50039 Fluorophore-2 ATG TTC ACT GAC ACA ATC TTA TGC AA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49361 0.40 uM
50035 0.40 uM
50036 0.40 uM
50037 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.