Protocol 36737: Probe Assay - Adgrg4<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

>chrX:56913179+56913269 91bp CAAATGTTTCACAGCAAATAGACC GGCAACAGTGTGGATGAATG

Mutant=  111 bp

Wild Type = 91 bp

X-Linked

Sequence

Wt Sequence (deletions in lower case; insertions with carrots ^g^ (g insertion):

CAAATGTTTCACAGCAAATAGACCTAACAACTTCTTCCCAA^g^actactggacttaacccacacaagacttcacattcatccacactgttgccagaaggcatggctgattctacaatcaacagtacagccatatcatatgctaatacagtaccctcacctctagcaacagtaagtgcagccaaagatttgaagacatctacaaccgagacagctacatttttaacagataccttgtttacttcaacagccacccctctgcctacccagactgtaacagaacactcatatctaggaaaaacaaggacatcaaaaagggtggaggccatggctactgagatctttcattcagctacagctactgatttaattgacacatctgtgtttactaagaattatactgtatctgaaacttcaacaactaagtccaagtcagctgttgggaagacaactttattcttgaatgaatcaacatccatagctccaacaccttgtcccaagcacaaatccacagatgttgctatactccatacctctaaatctgggcaagaatttcttgtatcttcagctgccagaactgtgtcttggtccacattagaagagacttcacctattactactgatgttgggattgtatcaaccttcccaccagagtctttgcttacctccacagcttctcctgtgagttctacatttcctgaaattcagttagcatctaccttgtcaacaactgattcagaaatggcatcaacagtccattcagtgttgcctatgcagaccatacctaccccaagaacagtgaaaccagagtcaggctctacaaatttccaagatgtcttttcacccagcatggaggatgctctatctgtctccatgcctaaagaaaccactttcatggccttttcttccattacctcctctccaatcaccagaactcaagatgaacagatagctattgatgctgagagtacacatctaaccataatccccggaaccaaatttgtccctacattggctgaagcttcactcttccctaccattgaagggcaagcatacactcaggatacacccacaacggatgagcccatgcttactttgacttctactaaatcaccttctacatataacgcatctgaatcagttctcacatctataacaattaaaagtgactatcagttcttcaccaatgagaccacttggacttctaagtcaggtcagaacctgttgacctctatgaatacaaccactatacccacattcacatctaataaaactctcacattaccattccaaggtaatgccactaatagagatcattcatccatgactactaacgtctcaccaattgaagcatccacggagagcaaagtgactaccagctctgatgctactacagccagttatacaacagctttattcaaaccaacatcccagtggcttagtcatttcacgagtgtttctggaatcacatctatagccagccagcctgaatctaaactcaccaccttattattgaaaagcaactctatgcccacagtagctacaaatgaatttccctcaataccaagtgagccagttgctccatcagtgaatacgtctaccctcactgacattaaaccaaatttttctacagagaagagcatttctgagacaatacaaatagaaacaaatggtgtgagctcatttggtgatactctggcccctctgcccatgtctgcaacaacacagagagtctataccactgtgacaaaagaaaccacttcccgtcatcctaaggtaaagtcaactatatcaacagtagctgaggcttctccattttcaacaatgctggaagtgacagatgaatcagaacaaatggtgacagcttctgtcactatttccccctttacagatatagaaaaattgactactgcattgagtaaagaaactgcaactgcagaggtgggagtgagttggctttcaaccaaattaaaggaaagcatgcctgagagttcacacaatggaaccacagaaagctttaactcgacccatacctacacagtagattggacttcagaaaaatccaagggtaactcagcttcatcacccaattctgcaagcacccaggcactacctgaacttccaagttcttcaacaatgaagacgatgggagtcactttctccactaattcttcacagaggacagctgcatctttgtctgctggtatcttgtctccacagactgcctctactcatgccttagtaactccccaactgcttacaca

 

This mutation deletes 5813 bp of ENSMUSE00000386381 (exon 3) and is predicted to cause a change of amino acid sequence after residue 254 and early truncation 9 amino acids later. There is a 1 bp insertion (G) at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49531 GAG AAC ATG GAG AAC ACA TGG A Mutant Reverse A
49532 CAA ATG TTT CAC AGC AAA TAG ACC Common A
49533 GGC AAC AGT GTG GAT GAA TG Wild type Reverse A
49534 Fluorophore-1 CTA CTG GAC TTA ACC CAC ACA AGA C Quencher-1 WT Probe
49535 Fluorophore-2 CAA CTT CTT CCC AAG TAC AGG TAT TT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49531 0.40 uM
49532 0.40 uM
49533 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.