Protocol 45696: Probe Assay - B4galt4<em1(IMPC)J> Alt 2
Version 3.0

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

>chr16:38574214+38574314 101bp GCTGTGTAAACCCCAGAGC AGGTCCGGCTTAAAAACGAG

Mutant=  99 bp

Wild Type = 101 bp

Sequence

Wt Sequence: gctgtgtaaaccccagagctccgtgccccggctccgattacAaccatgtgtcTggtgttgattgcagGAGGCCAGAGCAAGCTCGTTTTTAAGCCGGACCT

Mut Sequence:  gctgtgtaaaccccagagctccgtgccccgatatatatatatgtatacacacacacacacacacatatatatgtatatatgcatttgcctgcacgtatg

This mutation is a 807 bp deletion beginning at Chromosome 16 position 38,753,882 bp and ending after 38,754,688 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64984 AGG TCC GGC TTA AAA ACG AG Wild type Reverse A
64985 ATT CAC TTG CAT ACG TGC AG Mutant Reverse A
65943 Fluorophore-1 TCT GGT GTT GAT TGC AGG AG Quencher-1 WT Probe
66231 Fluorophore-2 CCG TGC CCC GAT ATA TAT ATA TGT ATA C Quencher-2 MUT Probe
66232 GCT GTG TAA ACC CCA GAG C Common A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64984 0.40 uM
64985 0.40 uM
66232 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.