Protocol 21639: Probe Assay - Mecp2<tm#Bird> Probe
Version 2.0

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=  95 bp

Wild Type = 103 bp

X-linked

Sequence

Wt Sequence: CCGTGACCGAGAGAGTTAGCTGActttacatagagcggattgCAAAGCAAACCAACAAGAATAAAGGCAGCTGTTGTCTCTTCTCCTTATGGGTAGGGCTCTG

 

Mutant Sequence: CCGTGACCGAGAGAGTTAGCTGtaaggatccaccggtcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtc

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
31298 GAC CAG GAT GGG CAC CAC Mutant Reverse A
32649 CCG TGA CCG AGA GAG TTA GC Common A
32650 CAG AGC CCT ACC CAT AAG GA Wild type Reverse A
32651 Fluorophore-1 ATG GTG AGC AAG GGC G Quencher-1 MUT Probe
32652 Fluorophore-2 CAT AGA GCG GAT TGC AAA GC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
31298 0.40 uM
32649 0.40 uM
32650 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 4.0 -- 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.