Protocol 36015: Probe Assay - Padi1<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

>chr4:140831117-140831207 91bp CCATTGTCCCAACCTAGAACC TGATCAGGGTCTGTCATCCTC

Mut= 87 bp

Wt= 91 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GCAGGACCTGAGCCAACCTTCTCTGGAGAGAATCCTGTCACCTGGGGCAAGTGGGGGAGGGGTGGGAGCCCCTGGACTTTGGCAAAAGTCCTGGCGCTCACTGGTTGTGGGACCAGACTGGTCATTCCCTCTCTACAGAGGATGAATAACGTCAAGGAAGGTCAAGCTGTCTAGGTCAGTAGGACAGACAGTAGCCCCAgtttgtctatctctctcgtatcctcagaaggtcctggagtcgccatacccaactttgtagccctattttctccttaaactgtgaggggctctggaacagtaccccaggtcacaattgccaaatagcttcaccttgccttctctcagggttgactctcttcccccaggtgaaggtgtcatactttgagtcacaggaggctgctgccctggcccacagtgtgctctacctaactgctgttggtgagtcccactccggcctgcctctggacacggtctggacatggatgtagtgacggtgactgaagctttcggggccttaggggggagcctcagctacccacctcacccctccgcttcttgggaacttcaaacatctccaggaccatagggattgagacccagccatgctcagagccaatagagcctagaaatgtcactgctatgagcacaatccacagtggcttcctcatgggagtcccattgtcccaacctagaaccatggagagtcataggcctctttcacccagagcccaCTGCAGCATCCTCTGAGGATGACAGACCCTGATCATAAGTCTCCAGGGGCTACATATAGTTAAGGGTACCCTGCATGTGACATAGAAACCCGGGGTCTCTAAGACATGGTCACTCAGTGCACATGGACAAGACAGGGACTTGTGTCCTTCCGACACCAGAATTGCCAATGTCGCTCTAGCCAGAGCCCCGAGGCCGACACACA

This mutation is a 532 bp deletion beginning at Chromosome 4 position 140,831,152 bp and ending after 140,831,683 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47580 CCA TTG TCC CAA CCT AGA ACC Wild type Forward A
47581 TGA TCA GGG TCT GTC ATC CTC Common A
47582 AAC GTC AAG GAA GGT CAA GC Mutant Forward A
47583 Fluorophore-1 AGT CAT AGG CCT CTT TCA CCC Quencher-1 WT Probe
47584 Fluorophore-2 AGA CAG TAG CCC CAC TGC A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47580 0.40 uM
47581 0.40 uM
47582 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.