Stock No: 008355
Protocol 21712: Probe Assay - Slc6a4<tm1Kpl> Probe Alternate1
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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=  92 bp

Wild Type = 125 bp

Sequence

Wt Sequence: gctcatacacgggtgtgctcacggctgcagtgataagtggcttagtcccagaagtacaaatggtgaggagtggtggaggacatggcccctggtacctgCagaggctctatgggaaggctgacaggtggatcCCgggggcgggggtggag

gggtgctggactcagaggcaggcagctggaatcttcctggggccagcctggaagggtaagagaggacccagatatagggctttaggcacctcccgaaggcgatgtttgcttctctcttcctctttagtctaccttggtgctccctttaagtgatggaag

gaactgtctcttccggctgg

 

Mutant Sequence: GGTTCAGTGGCCTGACCCCCTGGATCACGGGTGTGCTCATACACGGGTGTGCTCACGGCTGCAGTGATAAGTGGCTTAGTCCCAGAAGTACAAATGGTGAGGAGTGGTGAAGG

ACATGGCCCCTGGTACCTGCAGAGGCTCTATGGGAAGGCTGACAGGTGGATCCatcgattctagagatatcctcgagggcccctgcaggtcaattctaccgggtaggggaggcgcttttcccaaggcagtctggag

catgcgctttagcagccccgctgggcact

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
33082 TCT ATG GGA AGG CTG ACA GG Common A
33083 AGC CCT ATA TCT GGG TCC TCT Wild type Reverse A
33084 Fluorophore-1 CCC CTG CAG GTC AAT TCT ACC Quencher-1 MUT Probe
33085 Fluorophore-2 CTT CCT GGG GCC AGC CT Quencher-2 WT Probe
oIMR6592 GAA AAG CGC CTC CCC TAC Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
33082 0.40 uM
33083 0.40 uM
oIMR6592 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.