Protocol 35595: Probe Assay - Atpsckmt<em1(IMPC)J>Alternate1
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

>chr15:31606344+31606430 87bp TCATGAGGCTTCTGCAAAGAT GGTGGGACAACAGTTTAGCC 

Mut= 92 bp

Wt= 87 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

 ACACACACTCGAACACACACACACACACAGACACACAGACACACACACACACACACTCTCTCTCTCATACACACACACACACACTCTCTCTCTCTCTCTCTCACACACACACACACACACACACACACACACACACACACACACacGCAGCACGAGTCAGTGTGAGAAGGGCTGCTGAGAAGatgcagccttggctttctagtgtgtgctccaagtgtttctgctttcccccaatgatggctcaaatctaggaacgcctgaggaagaaagacaggcgggccctgttcttcctacgagtctggaatccgacagttcaaagaggaccagttggggattcttgattactggggtcgttggtggtgcactgctgacagtgtatgctgtggccacaccattcatcaccccggccctccggaaagtttgtttgccattcgtgcctgcaacttcgaagcaggttgaaaatgttgtcaggatgctgcgacacagaagaggacccctggtggacatcggcagtggcgatgggcggattgtgagttatcctaaaacattttaaggttaaactttaaccttaaaccttaataagagtggccttattcgaggaggtgtatcactgggggcacgctttgaggtttcaaaagcccaagccattcccagttagctgttaaactttctgctttatttttttcatgaggcttctgcaaagatttgccaattgtggcagttggaaaagtgtgtgtcttattttgcgtttggctaaactgttgtcccaccagcATGTGTTCAGAGTCCATTTTTCTGTGATTGATTTTTCCTCCCCTGGAGGAAAAGAACTCTAAACATGCCAGATAGAAAGGAGCAGTTAATTGCTAATTATAAAGTAGTCTATTAGGATTTTTGGTCCAGTAAAGGTGGCATGATCACA

This mutation is a 592 bp deletion beginning at Chromosome 15 position 31605842 bp and ending after 31606433 bp (GRCm38/mm10). In addition there is a 2 bp deletion [GT]36 bp after the 592 deletion.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45552 GTG TGA GAA GGG CTG CTG AG Mutant Forward A
45553 GGC ATG TTT AGA GTT CTT TTC CTC Mutant Reverse A
45555 Fluorophore-1 TGT GAT TGA TTT TTC CTC CCC Quencher-1 MUT Probe
46472 TCA TGA GGC TTC TGC AAA GAT Wild type Forward A
46473 GGT GGG ACA ACA GTT TAG CC Wild type Reverse A
46474 Fluorophore-2 ATT GTG GCA GTT GGA AAA GTG T Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45552 0.40 uM
45553 0.40 uM
46472 0.40 uM
46473 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.