Protocol 45918: Probe Assay - Slc13a5<em1/2(SLC13A5)Lutzy>
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

Mutant=  109 bp

Wild Type = 109 bp

Sequence

Wt Sequence:

AATCCGGTGGGACCGCGCGTCCCCCTTTTAAGCTCGTGAAGCTTCTAGCTGCCTCGCAATCTTTTCTCCCTCCAGTCAGTCTCCCTTTCACGCGATGGATTCGGCGAAGACTTGTGTGACCAAGTTCAAGTCCTTTGCGATTTTGCTCTTCACCCCGATCCTGATGCTTCCACTCGTCATTCTGATACCTGACAAG

Mutant Sequence:

CAAGGCCAAGGAGCTGCCAGGGAGTCAAGTGATTTTTGAAGGCCCCACTCTGGGGCAGCAGGAAGACCAAGAGCGGAAGAGGTTGTGTAAGGCCATGACCCTGTGCATCTGCTACGCGGCCAGCATCGGGGGCACCGCCACCCTGACCGGGACGGGACCCAACGTGGTGCTCCTGGGCCAGATGAACGAGTTGTTTCCTGACAGCAAGGACCTCGTGAACTTTGCTTCCTGGTTTGCATTTGCCTTTCCCAACATGCTGGTGATGCTGCTGTTCGCCT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
66789 CTT CTA GCT GCC TCG CAA TC Wild type Forward A
66790 AAG AGC AAA ATC GCA AAG GA Wild type Reverse A
66791 ACC CTG TGC ATC TGC TAC G Mutant Forward A
66792 TGC TGT CAG GAA ACA ACT CG Mutant Reverse A
66793 Fluorophore-1 TCA CGC GAT GGA TTC GGC Quencher-1 WT Probe
66794 Fluorophore-2 CCA GCA TCG GGG GCA CCG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
66789 0.40 uM
66790 0.40 uM
66791 0.40 uM
66792 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.