Protocol 45585: Probe Assay - Alg12<em1(IMPC)J> Alt1
Version 2.0

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

Mutant=  124 bp

Wild Type = 104 bp

  
>chr15:88811151-88811254 104bp GACTATATTTTCTGGAGTGCTGGT TAAAGGTGTGCGCCTCCAC

Sequence

Large deletion:  Mutant sequence with junction in uppercase

gtcctcatgacatacttcagcagccagccagccaggactgtgggactgaaaggaagtcagATaggtacttttaaaggttcgtcagaaataggtttgccctgggcggtggaggcgcacaccttta

 

This mutation is a 4434 bp deletion beginning at Chromosome 15 position 88,811,214 bp and ending after 88,815,647 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65217 GAC TAT ATT TTC TGG AGT GCT GGT Wild type Forward A
65218 TAA AGG TGT GCG CCT CCA C Common A
65219 GTC CTC ATG ACA TAC TTC AGC A Mutant Forward A
65220 Fluorophore-1 TTA ATT ACC AGG GTG ATA GGT ACT TTT A Quencher-1 WT Probe
65951 Fluorophore-2 ACT GTG GGA CTG AAA GGA AGT CAG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65217 0.40 uM
65218 0.40 uM
65219 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.