Stock No: 029239
Protocol 30633: Probe Assay - Mc1R floxed/floxed Probe Alternate1
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=  111 bp

Wild Type = 144 bp

Sequence

Wt Sequence:tggataccatggagagagtccagcagcaaccaagggtgggttgctaccaccactgcgtgctatcctgaccactatgaatagtcacatgcccagctgctggcCCacagaggctggagacagatctgccctgatgtggttcctggctccagggctagcatgtgctccacagcggaaatgagactcctcaagggaaggggtggggggt

 

Mutant Sequence:agacagcattggataccatggagagagtccagcagcaaccaagggtgggttgctaccaccactgcgtgctatcCTGACCACTATGAATAGTCACATGCCCAGCTGCTGGCCaattgtatcacgcgtataacttcgtatagcatacattatacgaagttatgccactagaggatccccgCACAGAGGCTGGAGACAGATCTGCCCTGATGTGGTTCCTGGCTCCAGGGCTAGCATGTGCTCCACAGCGGaaatgagactcctcaagggaaggggtgggg

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
34728 Fluorophore-1 AGT TAT GCC ACT AGA GGA TCC C Quencher-1 MUT Probe
34729 Fluorophore-2 TGC TGG CCC ACA GAG G Quencher-2 WT Probe
35055 GCT GGC CAA TTG TAT CAC G Mutant Forward A
35056 AGC ATT GGA TAC CAT GGA GAG Wild type Forward A
35057 GAA CCA CAT CAG GGC AGA Common A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
35055 0.40 uM
35056 0.40 uM
35057 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.