Protocol 34888: Probe Assay - Tacr3<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

>chr3:134861364+134861460 97bp CTTTGTTTGACAGGACTTTTCC ACTGTTCTGCAGTCAGACATTCG

Mut= 97 bp

Wt= 97 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TTGACACATGACTCTAGGTTACAATGTTTACAAGAGATTTGATAGGTGTGTGAAATGAAGACCTAGACATAAGGTGAAAATAAGGGCTGGTATTTGTATTGTGATTGAAAGTTCAACAAATTCCAAGTCCTGTATATTTCATCCTATAagattctttagcgtgtgtgtgtatttctctgcaggtaccacatcattgttatcatcctggtgtactgtttcccattgctcatcatgggtgtcacctacaccatcgttggaattactctctggggaggagagatcccaggagacacctgtgacaagtaccatgagcagcttaaggctaaacgaaaggtatggtcccatttaactgaccagtataactgtcccaaataactgacccctataactcacaagttggtgcagactgggccacttccaaggaggaaggcactgactgatgttagaaatatgttctcatatctcataatctgaatcctcctttgtttgacaggacttttcccttgcataacctctttgttctccctggcaaacctcagcccttggAAGGTCTGCGAATGTCTGACTGCAGAACAGTTACATCCTTAAGGATTAGCTGTTTTGTCAGTCCCTGTTGTGGCCCATTCTCAATTACTTTACCTGAAGTTCTTAAAGTGTCTCTTAAATGGAATCTAGCTGATTGTATTTCTGCA

This mutation is a 398 bp deletion beginning at Chromosome 3 position 134,861,032 bp and ending after 134,861,429 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44714 CTT TGT TTG ACA GGA CTT TTC C Wild type Forward A
44715 ACT GTT CTG CAG TCA GAC ATT CG Common A
44716 AAG GGC TGG TAT TTG TAT TGT G Mutant Forward A
44717 Fluorophore-1 CCC TGG CAA ACC TCA GC Quencher-1 WT Probe
44718 Fluorophore-2 AAG TTC AAC AAA TTC CAA GTC CTG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44714 0.40 uM
44715 0.40 uM
44716 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.