Protocol 36059: Probe Assay - Gtf2ird2<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

>chr5:134196569+134196669 101bp TGGAGAACAAAGCAGGGATCT GCTGCCATACTTGGCTCAG

Mut= 106 bp

Wt= 101 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CAGTGTCAGCCGTCACAAGAGGGATAGAGCCATCATGGCAGGAGAGCCAGCAATGGGCAGAGAAAGGGTGGGGCCAGGAGCGGGAAGCTGGCTGGTCACATTGCAGCCACACGCAGGAAGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAAGAGAGAACGCGCAAGCGCGTTAGTTCTTTGGGCTCTTGATGTATGTTCAAGCTACATGCTCCTGTGTCTATTGCCCTAGATGAAGCtggtggtagaaagtagttctgagagtcccagttaccatctcttacctcctgtcctaggtaaagccttggggacaacagccatggtccctgttccctacgagcagatgctacaggatgaggcagctgtggtggttcggggccttccggaaggcctggccttccagcacccagacaattacagccttgccaccctgaagtggatcctggagaacaaagcagggatctccttcgctgtaaagaggtaatctgctccggagCCTGACAGCCTGTTTAGAGAGAAACTGGCCTGAGCCAAGTATGGCAGCACAGGCCTGCAACCTCGGTACCGGGAGACTGAGACAGGAGAGTCACTGAAAGTTTAAGGCAAGCCCAAGTTACAGTGAGTTCAAGGACAGCCTGGGGTATATACCTAAATCCTGTCTTTTTTCTTTCTTTCTTTCTTTTTTTTTTTTTTAAGATTTATTTATTTCATGTATGTGGGTACAACTGTCGCTGTCTTCAGACACACCAGAAGAGGGCATCAGATC

This mutation is a 257 bp deletion beginning at Chromosome 5 position 134,196,365 bp and ending after 134,196,621 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47737 TGG AGA ACA AAG CAG GGA TCT Wild type Forward A
47738 GCT GCC ATA CTT GGC TCA G Common A
47739 TGG GCT CTT GAT GTA TGT TCA Mutant Forward A
47740 Fluorophore-1 AAA GAG GTA ATC TGC TCC GGA Quencher-1 WT Probe
47741 Fluorophore-2 CCC TAG ATG AAG CCC TGA CA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47737 0.40 uM
47738 0.40 uM
47739 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.