Protocol 36043: Probe Assay - P4ha3<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

>chr7:100293641+100293723 83bp GAGCTTGCTGCTGAGGGCTC CCCTCGCCTCCAAACACAG

Mut= 96 bp

Wt= 83 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AGCCCATGTGCTGCTCTTTGTACCCTCTCAGGTTACCTGGGCACGCTCACACACACACACACACACACACACACAAGCGCACACACTCACACACACATATACACACAACACACATGTATCCACTCATGCACACACATTCATGCATGCACATGTACACACACATGCACACACAAACACACACAATCTTTAACTAAAAGAATCCATTGTGTCCCAGGAGATGAGCTTGCTGCTGAGGGCTCTTTGagcaggtggtcaggtctcacatgagtgaaatgcccattgcctgtgtttggaggcgagggtcacagtctttctatttttacagctctgaaggatggctatgagaaggtagagcaagaccttcctgcctttgaggacctcgagggagcagcaagggccctgatgcggctgcaggatgtgtacatgctcaacgtgaagggcttggctcgaggcgtcttccagagggtcacgggctcctccatcaccgacctgtacagtcccaggcagctcttctctctcacagcggatgactgcttccaagttggcaaggtgacatgcaaagaagtccaactgctcgattctactttcttatggatggactgtgtctcagctgtctttaggtattccctttgcttgaggtaatgacAGGGTAGATCACTAAGACTGCATTGCAAAGTAGGAGGAGCTAAATTGAACCCCATCTTTTAGCTTCCCATGCTCTTGCCATAGGGATGAGAAGTCATCATATACTTAGGGCACAAGAAAAGCTTTTCTTTTCTTTCCAACAGGATATTGTGTATCCCAGGCTGGTGTTAAACTTGCCAGGGTCTTCCTGCCTCTACTTCTCCAGTGCTGGGATTACACCATGCCCATTTTTGCAGGGCTAGAAATCAAACCCAGGGTCTCATGTGT

This mutation is a 403 bp deletion beginning at Chromosome 7 position 100,293,665 bp and ending after 100,294,067 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47673 GAG CTT GCT GCT GAG GGC TC Common A
47674 CCC TCG CCT CCA AAC ACA G Wild type Reverse A
47675 GCA TGG GAA GCT AAA AGA TGG Mutant Reverse A
47676 Fluorophore-1 AGC AGG TGG TCA GGT CTC AC Quencher-1 WT Probe
47677 Fluorophore-2 CTG CAT TGC AAA GTA GGA GGA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47673 0.40 uM
47674 0.40 uM
47675 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.