Protocol 36275: Probe Assay - Spag6<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

>chr2:18715442+18715541 100bp GCATTTTCAGCACAGTTTGA TGGGAACTGCCTGGGATATG

Mut= 107 bp

Wt= 100 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TTAATATGATTTTTAGGTATATATCAGAGCATAGCTGCTATTCAGTGTAGTTCACTGAGGTCAGGAAAGTCTGCCATACTGAGAGGCTGAGGGGAAATAACAGAAAAGAGGTGGTCAGTCAACAGAGTTGGTTAATAGGCTAACATTCCAGAGTTCCCCTGAGTCAAACAGTACCTTTTAGTATCAAGCAGATGTAGAGAATCCAATTATTATATGAATTTGCAGAAAGTCCCATGTTTATATCTGATTTTATATAATTTCCAAATACATGCTTAAAAGTAAAGCATTTTCAGCACAGTTTGAAACTGACCTTGCTTCTAGGTTAGTTACTAACcagcggcaatgggagcgataaagactcaacatatcccaggcagttcccaccgtctgcgttccgaggcggttgtgctttccttcaatgccagttttgtgtcactttcaggctattttagattctaggtttggtctctttttccctgtagtgtgtctacaagaaggcagctgcgtttgtgctgcgagcggttggtaaacactctcctcagctggcgcaggccacagttgactgtggtgccctggactcactggtgatctgcttggaagattttgaccctggagtcaaggaggctgcagcctgggcacttgcatatattgcaaggcataatgcaggtactggggtgtgcttgaagatagactcaagtttaactttactagataagaacagttttgtttattatccattgatggagttttaaaagttgctggacattatgggagagctgtcttgaaggatagccacactgatttggatcacataattacaaaacacttctacatttgagttctagcagaagggaatctgaaggagacGGTGGTATGGTAGAGTGGGATTGGCATCCAGTAGCAAAGGTACATGCCTATAATCCCAGTATTCAAGAAGCAGACTCAGAAAGCTTAAAGTGAGCTCTGCAAGCCTGAGTTACACAGCCAGACACTGTCACAAAACCAAAACAACTAAGTCAGATGAAGAAAGTTCAGCCGACCAGGTAAATATATTCTTACTAACTTGTCAGAAGACTATTACTGGTTTCTCATGCTCTGAGCTCCTTGTCATTCACGACAGGACATTTCAAGCCAATCAAGGAGCCAGATCACATGGATGAATGGTGCGGTAAGATAATATACAGTTGTAGAAAATGTTTTATTACCCTGTGTTAGCAGGAAGGAGGAGGAAGAAAATAAGGGCAACACTTTTCTCAAAACTGAAATCCTGGTGTGGTCAACAAGACAAGACGGGGCTCTCCTGAGCGAAGACT

This mutation is a 533 bp deletion beginning at Chromosome 2 position 18,715,493 bp and ending after 18,716,025 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48272 GCA TTT TCA GCA CAG TTT GA Common A
48273 TGG GAA CTG CCT GGG ATA TG Wild type Reverse A
48274 GGA TTA TAG GCA TGT ACC TTT GC Mutant Reverse A
48275 Fluorophore-1 AAT GGG AGC GAT AAA GAC TCA A Quencher-1 WT Probe
48276 Fluorophore-2 ACT AAC GGT GGT ATG GTA GAG TGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48272 0.40 uM
48273 0.40 uM
48274 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.