Protocol 33532: Probe Assay - Rprd2<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

>chr3:95790391-95790529 139bp TTAAGGGTTTTTCGGTGCAT AACTGAAAAGCAGCCAGTGC

Mut= 139 bp

Wt= 139 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CCATTTTTTAATTGGGTTGTTTGTTTTTTTGGTGGTTAGCTTCTTGTTTTAATTTCTTTAATGGATTTTTTTTTTCTTTAGTGCCAAGTGTCTACAGAATGAGGTAGTCTACTTGGAAGAGTACCTTGAACACAAATTTAACCTCAAATAGTTAAGGGTTTTTCGGTGCATTTAATAAATTATCCATTAGttaatttcttattaccccctccatgtaattagtccatgtaaaagcaattacgtgaggctggagagatgctcaggttggaagcactggctgcttttcagttcctttctggcctccgtaggcactgcatgtgggtgatgtgcatacaagcaggcaaggcacccataagcacaaagtaacagcatggaggtttttaaaagttagctgtttgaaaacagtatatttgggaaaagttaatgacttttttttatctcttctctccagctacatatccccatcgtttgaatctcttttatcttgccaatgatgtcatacagaattgtaaacggaaaaatgcaatcatatttcgtgaatcatttgctgatgtgcttcctgaagcagctgctctagtgaagtaagtaaatctgtattgtacttggggattaattttttaatttttattttcttgtttttgcacttattaacaaaaacataaattgtgcatatttaaagacccaagtaaaatattgtgttattggacaagtgTAATGGTGTTTGTTTTTAATTTCAGCATCTGGGGAGCGGGGAAGGTGAAACTTTTAAATTTGAGACCTCGTTTCAAAAAAGCAAAAATGGGGTGAGCATAGGAGGTAGTAGCTCATTCTTATCTTTCTCTGGATTCATTCATAGTTTAATTTTTAGTGTTC

This mutation is a 522 bp deletion beginning at Chromosome 3 position 95,789,969 bp and ending after 95,790,490 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
41629 TTA AGG GTT TTT CGG TGC AT Common A
41630 AAC TGA AAA GCA GCC AGT GC Wild type Reverse A
41631 TAT GCT CAC CCC ATT TTT GC Mutant Reverse A
41632 Fluorophore-1 AGC AAT TAC GTG AGG CTG GA Quencher-1 WT Probe
41633 Fluorophore-2 ATC CAT TAG TAA TGG TGT TTG TTT TTA A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
41629 0.40 uM
41630 0.40 uM
41631 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.