Protocol 31526: Probe Assay - Gabrg3<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 transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

Mutant=  141 bp

Wild Type = 154 bp

>chr7:64578668-64578821 154bp GATCAAAAACTGTTCCCATTCT GCACTGTCCATGTGGAATGA

Sequence

Wt Sequence: GTGTATGTCTCCTTAAATGAAAATGGTGACAAGAAGCCATGTTTTCATTTTTTTCTTTGGAGTTGGGAGCCACTAGAACTTTGCTTTGAAACCGTAGGCTTTTTATCTACCACTGATATTGCTCATCCCATGATTTAGCTTTCTAGCAAGTTTGACATTAAATTATTTGTGCAGTGTCTCATATTTTATCTCATTTCTTTGCCATCAAAATCAACTTAAGTAACATCGAAAATCtttatagtcatctgttactggcaatttgcagtggggtagctgtcttgtttcccaaaagctcaataaatacgagtcaggatctctcatcctttgaagttttaagaagacaagatgaatttagttttaaatgcaaattaagtccccaagagaaatctgcccacctgagcagcatgggcctgtgttgttagtggctgaggcttctagacactgattggtttgtgctcttcatgtttcactgtaccataaagtactaacagatatgtatttttcccccacagtaaaaccaactgtgatcgatgtggacatttatgttaacagcattggtcctgtgtcatccataaacatggtaagaaacacctgtgtttaccttggtagctcctgtgcgtcttcatgcttacagtctccactggatttctgttgcagttttattaagcagatttaaagcccacgactgactcaagtgttaagtgccgtggtattgaaaatagtctttaagaatcagaagggacagtgtgacatcagcagtgagtcattgctctgaagatcaaaaactgttcccattcttacatacatagatagcacatgccattcaaagggaattatttagggttgtgtactcatTGGCTTTAGGCAACCTCACTAATATTAGGGATGCACGTAAGTCCAGTGAGAGAAGTCATTCCACATGGACAGTGCTTAGCTGCTAAGCACGCGATGCTGTACAAGAATGCATTACACCAAGCCACCTGACATGTCTGCCAGCTGTGGTGTGCCTCCTGATTAGATGATGTAGAAGTCCCATAGCAGGTCATT

 

Mutant Sequence:tttatagtcatctgttactggcaatttgcagtggggtagctgtcttgtttcccaaaagctcaataaatacgagtcaggatctctcatcctttgaagttttaagaagacaagatgaatttagttttaaatgcaaattaagtccccaagagaaatctgcccacctgagcagcatgggcctgtgttgttagtggctgaggcttctagacactgattggtttgtgctcttcatgtttcactgtaccataaagtactaacagatatgtatttttcccccacagtaaaaccaactgtgatcgatgtggacatttatgttaacagcattggtcctgtgtcatccataaacatggtaagaaacacctgtgtttaccttggtagctcctgtgcgtcttcatgcttacagtctccactggatttctgttgcagttttattaagcagatttaaagcccacgactgactcaagtgttaagtgccgtggtattgaaaatagtctttaagaatcagaagggacagtgtgacatcagcagtgagtcattgctctgaagatcaaaaactgttcccattcttacatacatagatagcacatgccattcaaagggaattatttagggttgtgtactcat

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37071 GCA CTG TCC ATG TGG AAT GA Mutant Reverse A
37072 GTG CAG TGT CTC ATA TTT TAT CTC A Mutant Forward A
37073 TCC CCC ACA GTA AAA CCA AC Wild type Forward A
37074 CAG TGG AGA CTG TAA GCA TGA AGA Wild type Reverse A
37075 Fluorophore-1 AGG GAT GCA CGT AAG TCC AGT GA Quencher-1 MUT Probe
37076 Fluorophore-2 TGG TCC TGT GTC ATC CAT AAA CAT G Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37071 0.40 uM
37072 0.40 uM
37073 0.40 uM
37074 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.