Protocol 35484: Probe Assay - Ak3<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

>chr19:29026989-29027077 89bp TATCTCCTGCAGCCTGGTG TCCATTTCCTATTCCAACCA

Mut= 89 bp

Wt= 89 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

CAGACTGGCCCTTAACTAACCCCACTAATGTGTAGCCAAGGCTTTGACCTCTGGGACCTCTTGCCTGCACTTCCTATGTGCTGGGATTACAGGCATGCCATCAACCCAGACTGGAGTTCAGTTTGGTTGGAGATTCTAGTAAGATGGCAGGCAGACCGTCTTCCCTTACTTCTCTACTCAGgcttgagtagagaaccctgggagggcaatgtgctgtctatacagaaatactgtcacatgcactcagaatgtttgcccaaatacctgagtactctacagcccagtcaagtagacaataaatttgatttcagttatattacggctctggtcaacatgcctggtgtcattccttgggctcaatttgcctaacactttttctttcttgcctgcctgtcccctctctcctcctcccctcatataaaggatttccaaggacacttccacaggcagaagccctggataaagtttatcagatagacacagtgataaatctcaacgtgccctttgaggtcattaaacagcgccttactgctcgctggatacatcctgccagtggtcgagtatacaacattgaattcaaccctcccaagactgtggtaagtgactgtagcgagctgagctgacatccactctaacaggcatagctgtgaacttctcatcctatctgaccaagactattgcacgggtcctatctcctgcagcctggtggtaagagacgtccccgtgctaatagGCTTGTTAGTCATTCAGAAGTGCTATGGTTGGAATAGGAAATGGATCCAACAGCTCAGTGTGGTGTCTGCTTAGTCTCCAGATGGTGGCATAATATTTGAGGTCCATCACAACTGGAAAAAGTGTGTCACTGGGACATCTGCAACCTATGAATGATCCTTAGCCCCTTCTCCACTCTGC

This mutation is a 552 bp deletion beginning at Chromosome 19 position 29,027,034 bp and ending after 29,027,585 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46182 TAT CTC CTG CAG CCT GGT G Wild type Forward A
46183 TCC ATT TCC TAT TCC AAC CA Common A
46184 AGT AAG ATG GCA GGC AGA CC Mutant Forward A
46185 Fluorophore-1 AGA GAC GTC CCC GTG CTA A Quencher-1 WT Probe
46186 Fluorophore-2 CCC TTA CTT CTC TAC TCA GGC TTG T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46182 0.40 uM
46183 0.40 uM
46184 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.