Protocol 20817: Probe Assay - Fanca<em1Dvs> 2 bp del
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= 126 bp

Wild Type = 128 bp

Mutation is a two bp deletion (aa)

Sequence

Wt Sequence:  TGTGCAGTGACATCTTCCTTCCTAACTCCTCTCCACGCaaAGTGCAGCGCAGCAGCTGCGATCCAGGCGGGGCTGGGTGCTGGGGAAGCTGACGCGGAGGCGAAActgacacacagacatcgttgcag

 

Mutant Sequence: TGTGCAGTGACATCTTCCTTCCTAACTCCTCTCCACGCAGTGCAGCGCAGCAGCTGCGATCCAGGCGGGGCTGGGTGCTGGGGAAGCTGACGCGGAGGCGAAActgacacacagacatcgttgcag

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
31097 CGA GCA GTG ACA TCT TCC TTC Forward A
31098 CTG CAA CGA TGT CTG TGT GTC Reverse A
31099 Fluorophore-1 TCT CCA CGC AAA GTG CAG Quencher-1 WT Probe
31100 Fluorophore-2 TCT CCA CGC AGT GCA GC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
31097 0.40 uM
31098 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.