Protocol 31525: Probe Assay - Asmt<em1(IMPC)J> Probe-alt2
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=  101 bp

Wild Type = 97 bp
>chrX:170674883+170674979 97bp CCACCCGTGACCTTTGAC GTACTGGCTCCGCCCTTC 
 
Asmt maps in the pseudoautosomal region of C57BL/6.   Males carry 2  copies, one on the X and one on the Y and can therefore be homozygous,  wild-type, or heterozygous on either the X or Y Chromosome.  Genetic  segregation and genotyping can be classified similar to that of  autosomal genes.
 

Sequence

Wt Sequence:

CCATTGTCCCGCCCCCTGACTTCCTGTCCCCGATGACGTCACCCTTGTCCCGCCCCCTGACTTCCTgcccccgatgacgtcactcctgccccacccctgacttc
ctgtccccgatgacgtcacccttgtcccgccccctgacttcctgtccccgcccccgcaggccccgcctacaccaactcccccctggcgtccaccttcctggtcgc
gggcagccccctgtctcagcgcagcctcctgctctacctggcgggcaccacctacctgtgctgggggcacctggcggacggcgtgaggtgggcggggcctggggg
ttgcgggggcggggccaggggcggggtcacacaggccacgcccatgagcaaccaggccctgcccaccaccctcagcttagtgaagccccgcccacggcgaggcca
agcccccacccgtgacctttgaccttcagtgggtgttagcggggtgggcgggGTCACGCGAATCGGTCCCGCCCCCTCCGCAGGGAAGGGCGGAGCCAGTACGC
GAGGGCCG
 
Mutant Sequence:
CCATTGTCCCGCCCCCTGACTTCCTGTCCCCGATGACGTCACCCTTGTCCCGCCCCCTGACTTCCtgTCACGCGAATCGGTCCCGCCCC
CTCCGCAGGGAAGGGCGGAGCCAGTACGCGAGGGCCG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
34418 GTA CTG GCT CCG CCC TTC Common A
36750 CCA CCC GTG ACC TTT GAC Wild type Forward A
36751 Fluorophore-1 TTC AGT GGG TGT TAG CGG G Quencher-1 WT Probe
37068 CTG ACT TCC TGT CCC CGA TG Mutant Forward A
37070 Fluorophore-2 CTG ACT TCC TGT CAC GCG AA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
34418 0.40 uM
36750 0.40 uM
37068 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.