Genotyping Protocols Database
Protocol 32405: Probe Assay - Il1rap KO (387 bp del) - Probe
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

Mut= 116 bp

Wt= 116 bp

Sequence

Wt Sequence (deletions in lower case):

CAAATTTGTTGTTGGTTCTGACAATAAAGCTTGGGAATATTctaaaacccttggaatggttcttagtctatagttatcacacattcacggtgattattattagaactcttcagggaagaagtaccctctcttactttttaaatttatgttgacaggatgggacttctgtggtatttgatgagtctgtccttctatgggatcctgcagagtcatgcttcgggtgagtatgtggtgctggataaatgactgaaattaatttaacatacaggatcctatatagtgaatttaaatgagcataatgttggttttgttccacagaattctgtagttgcatagaatcaagtgtctgaaagaaccatagaattaggggatgagagaaactagacaacttaggaaggggtctgatgagtataaacacagggacatctCTTCAACTTCATGCCCCACAGGCTAATTCTCTGTATTCTCATCTCAAGTACTGTGGAGGAAATGGCAGGGAGATT

387 bp deletion

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39019 CAA ATT TGT TGT TGG TTC TGA CA Common A
39020 AAT CTC CCT GCC ATT TCC TC Mutant Reverse A
39021 Fluorophore-1 TGG GAA TAT TCT TCA ACT TCA TGC Quencher-1 MUT Probe
39022 TCC CTG AAG AGT TCT AAT AAT AAT CAC Wild type Reverse A
39023 Fluorophore-2 TGG GAA TAT TCT AAA ACC CTT GG Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39019 0.40 uM
39020 0.40 uM
39022 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 4.0 -- 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.