Protocol 30409: Probe Assay - Tldc2<em1(IMPC)J> Probe
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

>chr2:156921239+156921333 95bp CACACTCAACCAGCCCATC ACTCTGGGACTTCCCCATCT

Mutant=  92 bp

Wild Type = 95 bp

Sequence

Wt Sequence: cacactcaaccagcccatccccatcacacgcttagtgacatcttagtgtgacgtgactgggaacgaGGagtcacaagatggggaagtcccagagt

 

Mutant Sequence: ttgaaagcaggaagctttggaggtttgattgttagcatttatttggcactgccaggatgcaggGGagtcacaagatggggaagtcccagagt

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
34543 CAC ACT CAA CCA GCC CAT C Wild type Forward A
34544 ACT CTG GGA CTT CCC CAT CT Common A
34545 TTG AAA GCA GGA AGC TTT GG Mutant Forward A
34546 Fluorophore-1 CAC ACG CTT AGT GAC ATC TTA GTG Quencher-1
34547 Fluorophore-2 TGG CAC TGC CAG GAT GC Quencher-2

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
34543 0.40 uM
34544 0.40 uM
34545 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.