Protocol 36171: Probe Assay - Zc3h7b<em1(IMPC)J>
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

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

>chr15:81768560+81768667 108bp GCCCTGAGTGGGTTGTTAGT GGACTTCCATTTTCCCTGCT

Mut= 111 bp

Wt= 108 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CCACGCTGGGCTTACCCTGGCCCTTTTCATCTTCTGCCCCCTTGAGCATCAGTTTTCCCCTCCACGTGGGACCAGGAGACAAGCCCTGCCCTTCTCCCAGGCTGACACAGTGCAAGCTCTCCTGAGCCAATGCCCTCCTCCAAAGGGAGCTGTAGTCTTACCCACAAGAGAGAATGTGTAGCCATTGGCCCTGAGTGGGTTGTTAGTGTCTGGTTGAGGGGTTGGCTGCTGTCACTGGGCctgccgctgactaactgagcagactcctctgggagagcagggaaaatggaagtccttgcatcagaactgagtgactccatgctgtccggtcacctcttacttcagccacagctgtctgaggcctactcttggtccacctcagccctgaagtgccagcctcagggctcctcatgggaccaacctgggttcaccctgtcctgtgtctctcttttcccactaggtcaacactacccctgaagcaagaggagtatgaggtgagtgtcgctgtggtgggtggatgcggtgagggCTGCTGTGCTCAGGGCAGAGGGCTGGGACTGAGCCAGGCTGGATGGAGACCCGCTTAGTCCCCTCGGACCTGCCTTGCGCAGGCTTTCCTGCTCAAGCTTGTGCAGAACCTGTTTGCCGAGGGCAACGACCTGTTCCGGGAGAAGGACTACAAGCAGGCTCTGGTGCAGTACATGGAAGGGCTGAACGTGGCTGACTACGCCGCCTCTGACCAGGTGGCTCTACCCCGGGAGCTGCTGTGCAAACTGCATGTCAACAGGGCAGCCT

This mutation is a 289 bp deletion beginning at Chromosome 15 position 81,768,613 bp and ending after 81,768,901 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48006 GCC CTG AGT GGG TTG TTA GT Common A
48007 GGA CTT CCA TTT TCC CTG CT Wild type Reverse A
48008 CTA AGC GGG TCT CCA TCC AG Mutant Reverse A
48009 Fluorophore-1 CCG CTG ACT AAC TGA GCA GAC Quencher-1 WT Probe
48010 Fluorophore-2 ACT GGG CCT GCT GTG CT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48006 0.40 uM
48007 0.40 uM
48008 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.