Protocol 34746: Probe Assay - Tmem164<em1(IMPC)J>
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

>chrX:142770869+142770978 110bp CATGTGGGCTGTTTTCCAC TGCATCTGTAACCTGGAGGA

Mutant=  85 bp

Wild Type = 110 bp

Sequence

Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion):

 

A 340 bp deletion beginning at Chromosome X position 142,770,892 bp and ending after 142,771,231 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44376 CAT GTG GGC TGT TTT CCA C Common A
44377 TGC ATC TGT AAC CTG GAG GA Wild type Reverse A
44378 TTG CAA AGC GTA CAA AGG TTC Mutant Reverse A
44379 Fluorophore-1 TGT CTG AGC CCT TCG TTC TT Quencher-1 MUT Probe
44380 Fluorophore-2 CTC GCA CAT GGC TTA GCT C Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44376 0.40 uM
44377 0.40 uM
44378 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.