Protocol 36274: Probe Assay - Frmd3<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

>chr4:74119706+74119805 100bp TGGTTGGTTCTTTCATGTGC GGATGAGCTGAAACGTCATAAAG

Mut= 91 bp

Wt= 100 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

AAATTCTTTCATGTATCTCAAACCTAAACACAGCTTTGGGCAGATTGGCTTTTACGTGAGAGTGGAAAGTGTCAGAGGAGAAAATCTTCCTTGATCTGTGATCTGGAGAGTTGGAAGGGAAGGTGAGTTGTGATGTAGAGTGATGAGCTTCTTCTTGCTTTGGGTTGTATTGTACATTTGCTCTGGATATCCTGTTAAACATGTTTTTTGTTTTTGTCACCATCTTGAGCATTCTATTGGCTTCTTTCCTACAACCTCCTTCCCTTCCCTTCTGGTTGGTTCTTTCATGTGCTGAGGTTCCTATGTGTGTTTGCCTCTGTGatcttttgccgcttatctcatgccttttctttatgacgtttcagctcatccaccatacaccatgtgctttagagtgaaattctacccacatgaacccttgaagattaaagaagagctcacaaggtatttttcctttttgcatatttactccaccgcttgcattcaggtagagcctttgctatcttataggctagacattcctgttatcgatttgagaacacctccagGAAGTAGGCTGTGGAGTCAGCATTGTGTTCCTCATATTCTTGTTGAATTTGGGATACTTTATAGAGAACCTTGTTGAGAAAGTATTATCTGTACAAGAAAGCAATGGTTATAAAAGGGGAGCTCTCTAAAACATTTGACAATGGAATGCTAACAAAAGCAGAAGATGACTGTCTTTTGGGTATGGGCAAATGCAGAGAGCAGTCCTAGTCTGTCCTTTTGGGTCTAAGACTTTCTGGAAGAACAAGATTACTGGACAGAGAGGACAATTAGCTATTTTGCTTTCCACTTGCTACATGACAACTACCAAGCAGATGAACTCTCCAACTTTCTTCTCTTTCTCTCTCTCTC

This mutation is a 227 bp deletion beginning at Chromosome 4 position 74,119,755 bp and ending after 74,119,981 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48267 TGG TTG GTT CTT TCA TGT GC Common A
48268 GGA TGA GCT GAA ACG TCA TAA AG Wild type Reverse A
48269 CAA GAA TAT GAG GAA CAC AAT GCT G Mutant Reverse A
48270 Fluorophore-1 ATC TTT TGC CGC TTA TCT CAT G Quencher-1 WT Probe
48271 Fluorophore-2 TCT GTG GAA GTA GGC TGT GGA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48267 0.40 uM
48268 0.40 uM
48269 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.