Protocol 33039: Probe Assay - Gt(ROSA)26Sor<tm2(CAG-luc)Tyj> Probe Alternate1
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=  117 bp

Wild Type = 126 bp

>chr6:113025997-113026122 126bp GAGTTCTCTGCTGCCTCCTG AGCCTGCCCAGAAGACTCC

Sequence

Wt Sequence: caatacctttctgggagttctctgctgcctcctggcttctgaggaccgccctgggcctgggagaatcccttccccctcttccctcgtgatctgcaactccagtctTTCTagaagatgggcgggagtcttctgggcaggcttaaaggctaacctggtgtgtgggcgttgtcctgcaggggaattgaacaggtgtaaaattggagggacaagacttcccacagattttcggttttgtcgggaagttttttaatag

 

Mutant Sequence: AAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGatcgccgtgactagaggcaataaaaagacagaataagacgcacgggtgttgggtcgtttgttcgaattcaCTAGAAGATGGGCGGGAGTCTTCTGGGCAGGCTTA

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
24493 AGC CTG CCC AGA AGA CTC C Common A
33533 Fluorophore-1 CAA CTC CAG TCT TTC TAG AAG ATG G Quencher-1 WT Probe
39497 AGA AGG GCG GAA AGA TCG Mutant Forward A
39499 Fluorophore-2 ACG CAC GGG TGT TGG GTC Quencher-2 MUT Probe
oIMR4981 GAG TTC TCT GCT GCC TCC TG Wild type Forward A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
24493 0.40 uM
39497 0.40 uM
oIMR4981 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.