Protocol 31606: Probe Assay - Gpr137<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

Mutant=  142 bp

Wild Type = 148 bp

>chr19:6939718-6939865 148bp AGGTGTGGCCATCTGAGTTC CAGTGAAGCACCCACGAAG

Sequence

Wt Sequence (deletion in lower case)

CATCTGCAGAAAGAGATGAGCAGAGTTTAGGTGTGGCCATCTGAGTTCTAACTGACCCTGGGGTCTCCTGAGgagtcagggttgctggtgagggccgaggggatccgtggtctggggctgactttgctgtgggctgcaggctggctgtccgaggggccttcgtgggtgcttcactgctctttttgctggtgaatgtactgtgtgcagtgctgtctcgccagcgccaggcacagccctgggtccttctgctggtacgtgtcctggtgagcgactctctcttcgtcatctgtgccctctcgcttgctgcctgcctctgccttgtggcccgtcgagccccttccaccagcatctacttagaggccaaggtagagccaccaaccttgatgtagcacccttgggggtccttggacagagttctttaggatgtaaaggaatataggagccctcctctccatgaagatcctctattgtcatctctgccaggggaccagcgtgtgtcaggcagctgctatagggggtgccatggtccttctctatgccagccgggcctgttacaacctggcagctctggccttggcccctcggagccggctagatgccttcgattacgactggtacaacgtatctgaccaggtaggcattggtcctgtttgtcccttctttggtaactataataactagcctccagctggccagggccctgccagcagcctctgtggtggcagaatgtcctcagcccctgcttttccccaggcagatctggtgaatgacctagggaacaaaggctacctggtgtttggtctcatcctcttcgtttgggaactgctgcccaccacattgctggtgggcttcttccgggtacaccggcccccacaggatctggtagggacccccccccaaccacctcttggtcCAGTATAGAGGGCTGGACCCTGGGAATTCTGGTCTGGGGTCCTGGGACAGTTACGGCAGCAGAAGAAAATATCCATAGTACCGACTTCTTTCCCCATACCTTAC

841 bp deletion beginning at Chromosome 19 negative strand position 6,939,821 bp and ending after 6,938,981 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37266 AGG TGT GGC CAT CTG AGT TC Common A
37267 TAT GGG GAA AGA AGT CGG TAC T Mutant Reverse A
37268 CAG TGA AGC ACC CAC GAA G Wild type Reverse A
37269 Fluorophore-1 TCC TGG GAC AGT TAC GGC Quencher-1 MUT Probe
37270 Fluorophore-2 CTG ACT TTG CTG TGG GCT G Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37266 0.40 uM
37267 0.40 uM
37268 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.