Protocol 33179: Probe Assay - Ankrd52<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

>chr10:128380453+128380544 92bp CCCTCAATGTTGCAAACACA GCCCTCAACCTACCAAAGTACA

Mut= 87 bp

Wt= 92 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case; and insertion with carrots ^g^ (GTCC insertion):

 GGCAGAGGCAGGCGGATTTCTGAGTTCGAGGCCAGCCTGGTCTACAGAGTGAGTTCCAGGACAGCCAGGGCTACATAGAGAAACCCTGTCTCGAAAAAACAAACAAACAAACAAAAGAACAGAGGTATCAGCTGTCACCCTCAATGTTGCAAACACAAAGGGGGAAGATTCTGAGTGCACCTCTCTT^ttcaggaggtcagaggcatctgtactttggtaggttgagggcatgaacctgttccgtgtcttttatatcctcatatagacagtgaacctgctcctgaataagggagccagcctgaatgtctgcgataaaaaggaacggcagccgctgcactgggcagcttttctaggtgagagacactgtaggaaaagggagaatttgattgttaggatccccagggcaagtttcaccctttggggg^TGGAGCCTGGAGCCTTTGGGGGAGGAGCCTAGAGCCTTTGGGGAGGGAGCCTGGGGCCtttgggggtggagcttagggcctttgggtttacCCTCCACCTGCTTGCTTCTCCTTCCTTCCTTTCCTTCTTCCTTCCTTTCCTTTCCTCCCTCCTTCCGTTTCTCATTCCTTCCTTTTTTTCTTCATAGGGCATTTAGAGGTCCTGAAACTGCTGGTGGCACGTGGAGCA

This mutation is a 237 bp deletion beginning at Chromosome 10 position 128,380,503 bp and ending after 128,380,739 bp (GRCm38/mm10). In addition, there is a 33 bp deletion (TTTGGGGGTGGAGCTTAGGGCCTTTGGGTTTAC) 58 bp after the exon deletion and a 4 bp insertion (GTCC) at the exon deletion site, that will not alter the results of the exon deletion. 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40836 CCC TCA ATG TTG CAA ACA CA Common A
40837 GCC CTC AAC CTA CCA AAG TAC A Wild type Reverse A
40839 TCT AGG CTC CTC CCC CAA A Mutant Reverse A
40841 Fluorophore-1 TTC AGG AGG TCA GAG GCA TC Quencher-1 WT Probe
40842 Fluorophore-2 CCT CTC TTG TCC TGG AGC CT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40836 0.40 uM
40837 0.40 uM
40839 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.