Protocol 33017: Probe Assay - Mypn<em1(IMPC)J>-Alternate 1
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

>chr10:63169613-63169703 91bp TCTGACTCAAAGCTTGTATGTAGGT CCAAAACGAACACATGAAACTG

Mut= 109 bp

Wt= 91 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case, insertions in^^):

 

CACCATTGTCATTTTCAGCAGGAGTCACTTCTCAATTCCCGGAGTAAAATATCAGTGTGGTTTGGATACTGCCGACTCATCTCGCTGTTACACTCAAACAATACCGAACCAAAGG^T^TTTACTTCGCATTCTGACTCAAAGCTTGTATGTAGGTC^ACCAAGGCA^tcctcaggaaacagctggagccatctctactcttgatattattcagtttcatgtgttcgttttggttgtagtttgttgtagttgttttgaggcagggcctcctatagctcaggctgacctccaatatgtaggctaggatgaccttgaacctctgacgctgccacctccacctcccctgtaaatcccaccatccctgacaactctccggtttctgctgtgtctgatggcgatatactcgtccttgcaggtggtattgtgaaggcaaggagcttgaaaactctccagatatccacattgtgcaggcaggaaaccttcactcactgaccatcgccgaggccttcgaagaggacacaggacgatactcatgctttgcttccaacatttatgggacagattcaacttccgctgagatttacatagaaggtaaagctaaatacttttggagtgtgacatgttctaactatctgtagttacgtaaacatttgtaggggggggcttttctacccatctagacactggggatactagtcatgaaccaagtagacaaaactcttcagcggatgtgccgatgaattcccgTTATAATGGGAGAAACTCCAAACAAATGaGATAAACCATGATAAATACATAACATTGCAAAAGAAATAGGAAAAGAGATGATCAATAAAGAGCTCACTCACCAGGTGACATTTGAGGGAAAAGCTGGTGGCCACAGGAGAG

This mutation is a 579 bp deletion beginning at Chromosome 10 position 63,169,099 bp and ending after 63,169,677 bp (GRCm38/mm10). In addition, there is a 9 bp insertion (TGCCTTGGT) at the deletion site and a single bp insertion (A) 38 bp after the exon deletion 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
39524 TCT GAC TCA AAG CTT GTA TGT AGG T Common A
39525 CCA AAA CGA ACA CAT GAA ACT G Wild type Reverse A
39526 CTT TTC CTA TTT CTT TTG CAA TG Mutant Reverse A
39527 Fluorophore-1 CAG GAA ACA GCT GGA GCC Quencher-1 WT Probe
40474 Fluorophore-2 ACC AAG GCA TTA TAA TGG GAG A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39524 0.40 uM
39525 0.40 uM
39526 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.