Protocol 36034: Probe Assay - Pcnx4<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

>chr12:72556493+72556589 97bp GCCCACGAAAGCTATTTACC GGGGACACTGAAGCAAAACA

Mut= 95 bp

Wt= 97 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AAGCATCTACATCTTTGGGATTTTCCGAAACCCTTTCTATCCGAAGGATATACACGCTGTGTCTGTATTCTATGACAAGCAAAACAAGCTCCTGAAGATTGGTGCTGTCAGATGGATTTTGCTAACTCTAGGTAGGAAGGTTCTATTTATCAACTCTTACAAACTGTAGTTAGGGAAGACTGCTCACATTTATGAAATAGACATAATCTTTATGTATGATTTTTTTGTTGTTGTTTATTAGCTTTTCAGCTAATTGCTTAAATGTGCCCACGAAAGCTATTTACCAAGATAAActgtggtaggagcagtttctgcagtagtcagactcaccatgcattggtctgttttgcttcagtgtcccctttggcaatggtggccttccttgcgctggacagctccttacacgggctccactctgtgtctgtctccattggattcaccagggctttcagaatggtaattccagtgtgcactgtgaagcacggccggtgGCTGAGTGTAACTGTGGTTTTTATTAACAGTTTGCTTAGATTGCTGAATTCTACCTGAAAGTGGATCATTTGTATACTGTAGGTGTGGCAGAATACAGAAACTGCTTTACTGGAGACTGTGGTTGTATCAGCAGTGCACATGGTGTCCAGCACAGACTGCTGGTGGAACAGGAGCCTTGATACAGGAATCAGGCTGTTATTGGTAAGTGCATTTTTAACAGCTCTGTTGAGAGATTTTTTTTTTTTAACTCACAATCAAACTCATCCAATTGAAGGCTTCTT

This mutation is a 198 bp deletion beginning at Chromosome 12 position 72,556,521 bp and ending after 72,556,718 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47640 GCC CAC GAA AGC TAT TTA CC Common A
47641 GGG GAC ACT GAA GCA AAA CA Wild type Reverse A
47642 GAT CCA CTT TCA GGT AGA ATT CAG Mutant Reverse A
47643 Fluorophore-1 CAG TAG TCA GAC TCA CCA TGC AT Quencher-1 WT Probe
47644 Fluorophore-2 AAA GCT GAG TGT AAC TGT GGT TTT TA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47640 0.40 uM
47641 0.40 uM
47642 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.