Protocol 34200: Probe Assay - Cyp4f39<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

>chr17:32470591+32470715 125bp CAGGAGCTCTATCCCTGAGC CTCTCTTCCCTCTAGAACACCA

Mut= 120 bp

Wt= 125 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TCTTTGGAATCCAGGTTCTCACACATATGAGGCAATATCTCACACATTCTACCCCACAGGCATGTCCCCAAACCCTCACTGGGGGATTCTGGTCAGGAGCTCTATCCCTGAGCCACGCCCCAGCCCCATTGTTCTTTCTTGTTTTAGAGtggggagcaactcagggaagctcagcaaggcaccatgtctttgcttttggtgttctagagggaagagagagtagtgagagggagaggagggggagggagggagggacagactaagccgagctgtcaggtcctcattgcccctttctatctgccacagtatcttcccaatgagaagggccttcaaaacgagaagaaggtgctggacaccatgcaccacatcatcctggcatgggttggacctttcttgcctctcctggtgcttgttcatcctgattacatcaagcctgtgttgggtgcctcaggtaggcaggttggaccgcagagggatgagtcaacttgaaactcccagaacccttctagggttacagacatgtgggtgccccacaggaagtgtgccttgagtaagtgtttgagggatggatagaagttcaccaaacTGGGGCCGGCAAAGCAGGTTGTGAGAACATGGACAATTGTGGGGTGGTGTGCAAGAGTTTCAGGAACATTGTAAGTCTCAGGCGCACCTGCTGGAGTCACATCTAAGCAACTGACTTATTCAAGGTTTTGGTAAGTAATTGTCGTTCAATTGTAGGCCATGACAGTCCAT

This mutation is a 437 bp deletion beginning at Chromosome 17 position 32,470,647 bp and ending after 32,471,083 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43042 CAG GAG CTC TAT CCC TGA GC Common A
43043 CTC TCT TCC CTC TAG AAC ACC A Wild type Reverse A
43044 CCT GAA ACT CTT GCA CAC CAC Mutant Reverse A
43045 Fluorophore-1 AAC TCA GGG AAG CTC AGC AA Quencher-1 WT Probe
43046 Fluorophore-2 TTA GAG TGG GGC CGG C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
43042 0.40 uM
43043 0.40 uM
43044 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.