Protocol 45411: Probe Assay - Pla1a<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

>chr16:38415643-38415752 110bp TGGCTGGAATCTGCTATGTG CCAGATGTGGAACCAGCAC

Mutant=  83 bp

Wild Type = 110 bp

Sequence

Wt Sequence: tggctggaatctgctatgtgttgtgagttgtacatgcaAcgagaggccctttcagataatttacatgtgcggatgctggaaagctccacgtgtgctggttccacatctgg

Mut Sequence: tggctggaatctgctatgtgttgtgagttgtacatgcaAGtcacaagacatggtattgcacaaaaataaggaatgctgactgt

A 544 bp deletion beginning at Chromosome 16 position 38,415,170 bp and ending after 38,415,713 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65536 TGG CTG GAA TCT GCT ATG TG Common A
65537 ACA GTC AGC ATT CCT TAT TTT TGT G Mutant Reverse A
65538 CCA GAT GTG GAA CCA GCA C Wild type Reverse A
65539 Fluorophore-1 TAC ATG CAA GTC ACA AGA CAT GGT Quencher-1 MUT Probe
65540 Fluorophore-2 AGG CCC TTT CAG ATA ATT TAC ATG T Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65536 0.40 uM
65537 0.40 uM
65538 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.