Stock No: 004424
Protocol 20106: Probe Assay - F8<tm1Kaz> Probe
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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= 112  bp

Wild Type = 110 bp

Sequence

Wt Sequence:

 

cctattgccctcaaagaccataaggaggctttggatcccctggaactggagttaaaaacagttgtgagctgaaatgtgggtactgggaataaaacacagatcttctggaagag

agccagtgctcttaaccattgagctatcttttccagcctgattTTTAtcttctatggtaagcattttggtatttatcctaagcacagtgagaattttaagcaagggaagtgatatcacttttcta

 

Mutant Sequence:

 

CTTTCCCGCCCTCCCTTGCGCTACGGGGCCGCCCGCACCGGCCTACACGGAGCGCGCGCGGCGGAGTTGTTGACCT

CGAGCggtaTCTTCTATGGTAAGCATTTTGGTATTTATCCTAAGCACAGTGAGAATTTTAAGCAAGGGAAGTGATATCACTTTTCTA

TGTTAGAAATATTACTCTGGAATCTACTACATAGAATGAGTAGTACTTTGGCATGCAAGTTGATAAGAGAGAACTTACACTAATACAATTAATGCCCTCCTGGGAACTCTAGGAAAGAGATATGCACTCG

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
29854 CCC TTG CTT AAA ATT CTC ACT G Common A
29855 CCG CAC CGG CCT ACA Mutant Forward A
29856 GAG CAG CCA GTG CTC TTA ACC Wild type Forward A
29857 Fluorophore-1 TGA GCT ATC TTT TCC AGC CTG A Quencher-1 WT Probe
29858 Fluorophore-2 CGG CGG AGT TGT TGA CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
29854 0.40 uM
29855 0.40 uM
29856 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.