Protocol 30408: Probe Assay - Tmem171<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

>chr13:99458128-99458219 92bp AGGCTTGGGTGTATCTGGTG CTCGTCACTCAGCCTCCAGT

Mutant=  90 bp

Wild Type = 92 bp

Sequence

Wt Sequence: ggcttgggtgtatctggtggcatgtttgagatcccgcctcgCTgctcacactcagccgggaaaccttgggcactggaggctgagtgacgag

 

Mutant Sequence: ccaacccagcctgttttacttgacatggaacaggtccacttctccatccccgcgCTgctcacactcagccgggaaaccttgggcactggaggctgagtgacgag

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
34538 AGG CTT GGG TGT ATC TGG TG Wild type Forward A
34539 CTC GTC ACT CAG CCT CCA GT Common A
34540 CCA ACC CAG CCT GTT TTA CT Mutant Forward A
34541 Fluorophore-1 CAT GTT TGA GAT CCC GCC Quencher-1 WT Probe
34542 Fluorophore-2 CAT GGA ACA GGT CCA CTT CTC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
34538 0.40 uM
34539 0.40 uM
34540 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.