Protocol 33047: Probe Assay - Frmd4a<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

>chr2:4472959+4473068 110bp CATCCAAGCCTCTCTTTGGA TCACGTCATCATCTTGGGTCT

Mut= 96 bp

Wt= 110 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GCCTCTATTTCTCGTGGAACCCCATGAAGTACTCATGCACCATGACAACCAGTTTTATGCTGCTTGGATGATTAAACACAGGGCTTTGTGCGTGCCAGGCAAGAACCTTCCTTCCAACTGAACCACATCCAAGCCTCTCTTTGGATTTTACAAAAGTCATTTGTGTTTCTGCAGTGTGCCTGAGTTCtcaggcatttcactgagaacatgctagagacccaagatgatgacgtgacttagcaaaagtctctcgtgagttaatcttcccttctgtgttcttcttccagcccaagcttttggccaaggagcttcttgacctcgtggcctctcatttcaacctgaaggagaaggagtactttggaatagcattcacagatgagacgtaagtgctgtccagagcccacagaccccaaacaaaagagcaggatgtcaaattaccctctctctgctgtgcacagcaaaatcaggctgcaggccacttgggtcccatgggggctacagggtcagtccaagttcaccatctgaatttcaaaagccggtgATCATGCCTGGAGTGGCCAAAGGATATAATTAGATAGATGCTAATATTATGAAATTATTAGGTAATGTTCTTTAACCAAGCAGATTTGTGTTCAGATGAATGTCTAAAATGTTTATTTTTTTTAAAGAGTAGATCTGTGTGGGTTTAGTCTTTATTAAGGTTAAATGAAAACTTCTGGAAAGT

This mutation is a 354 bp deletion beginning at Chromosome 2 position 4,473,021 bp and ending after 4,473,374 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40535 CAT CCA AGC CTC TCT TTG GA Common A
40536 TCA CGT CAT CAT CTT GGG TCT Wild type Reverse A
40537 TCT AAT TAT ATC CTT TGG CCA CTC C Mutant Reverse A
40538 Fluorophore-1 CAG GCA TTT CAC TGA GAA CAT G Quencher-1 WT Probe
40540 Fluorophore-2 AGT GTG CCT GAG TTC ATC ATG C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40535 0.40 uM
40536 0.40 uM
40537 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.