Protocol 32133: Probe Assay - Coq3<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

>chr4:21893103+21893270 168bp AGGGTTGGAAAGCTTCAAGG GGAAACAAGGCACTCTCCAG

Mut= 170 bp

Wt= 168 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence: (deleted region in lower case)

 

GCTCATTGAGTGAGAGCATTTGCTGGTTTTGGAGAGGACTAGTTTAGTTCCCAAGACCCATGTCAGATGGCTTGAAAGTGCCTGTAGCTCCAGCTCCATGGGATCTAGCATTCTTTTCTGGCCTCCACAGGCACTTGCACACACTCACACACTCCACAGAGTGACAGATTTTACATATTCACTACTTAATTTTGATAATTTTAATTTTTAGATGGAAAAAAGTAAAACCTTTATTTTATTTTTTAATTAAATTCCAAATTAATGCctttgttatcccaaagaaactcctttgcttatcatttgcaatacaccatgagaatatcataaaaattcttcaactgtgttggtatcaggactcacatgctcctctctgcttttaacctttgttttcagcttactcagggacgcagctcactaggaatctacagattaagccgtgggaactcggtgaacacgggaccatgtgctttagatcctacaggatggctctctcctgtttgagcagagtaaaaacttacaggtgagtctgtcaattattctagtagagatttacatattttgtgtaacagcagtttagttttgattttgtttaataacctttgccagtatgttaaagcttatgtggtctaatttaagtcgcttttacttgtttaaataattttaaataattattttttaataatttttagggttggaaagcttcaaggtcacctaaaagttagaagggaaatatagtacattagccatgaatggtatttatttacgttcagagacaattattatttttaatgaaaattagacacatttctttcaggttcaccagcCTCCTTCCACCCTGGAGAGTGCCTTGTTTCCCTTAATAACCCTTATTACTAGTGTTCTATTGCTGTGAAGAGACACCATTACCATGGCAAGTCTTATAAAGGACACCAGTTAACTGGGGCTGGCTGACAGGTTCAGAGGTTTAGTTCATTGTCACCATGGTGGAAAGCATGGCAGCATGCAGGCAACATGGTACTGGGAAAGGAGCTGAGAGGTCTACATCTGGATAGG

This mutation is a 559 bp deletion beginning at Chromosome 4 positive strand position 21,892,681 bp and ending after 21,893,239 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38424 AGG GTT GGA AAG CTT CAA GG Wild type Forward A
38425 GGA AAC AAG GCA CTC TCC AG Common A
38426 ACA GGC ACT TGC ACA CAC TC Mutant Forward A
38427 Fluorophore-1 AGA AGG GAA ATA TAG TAC ATT AGC CAT G Quencher-1 WT Probe
38428 Fluorophore-2 CAC TCC ACA GAG TGA CAG ATT TTA C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38424 0.40 uM
38425 0.40 uM
38426 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.