Protocol 33716: Probe Assay - Palmd<em1(IMPC)J>Alternate1
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

>chr3:116948069-116948166 98bp GCCAAGTCAGTGGGAAAAAC ATTTGCCAGCAATTCCCAAG

Mut= 106 bp

Wt= 98 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case, insertion with carrots ^g^ (AT insertion):

CCACCTTATTTTGACAGATTTTTACATGATCATAATAGTTATTTGTAATTATTAAAATATTGATTTGGTTTCTTTTTAAAGAAGAAAGGGGTCAACAAGTTTACAAGGAAGGGAAAATGCCAGATTTATTGAGCCAAGTCAGTGGGAAAAACTAATCTCTGTTGATATG^cccgctgtcgccatgcataagacttgaaagcaataggagatcttgggaattgctggcaaatataagaacatacgtttatattccctgacatcagaaaaaagccctgagggagaaatggctcctggatggaatcggcagtggaaaagaacacgaagagatgaagaagcaaaatcagcaagaccagcaccagacccaggttctagagcaaagcatcctcaggtacggccctcactggggtacttctcaaaccgagctccttccctaaa^AGGGAGCTGGGgtGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCACACGCATGCTCTCTCAGGTATACTCACACATGTAGAAGCCAGATATGAATGCTGGCATGTCTTCCTCAGTTGCTTCTCCCAGATTTTTAATATTTTTATTAATTCTTGAGAACTCCA

This mutation is a 266 bp deletion beginning at Chromosome 3 position 116,947,864 bp and ending after 116,948,129 bp (GRCm38/mm10).In addition, there is an AT insertion at the deletion site as well as a 2 bp deletion GT 11 bp after the 266 bp deletion that will not alter the results of the deletion.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40546 GCC AAG TCA GTG GGA AAA AC Common A
40547 ATT TGC CAG CAA TTC CCA AG Wild type Reverse A
40548 CTG AGA GAG CAT GCG TGT G Mutant Reverse A
40549 Fluorophore-1 CTG TCG CCA TGC ATA AGA CTT Quencher-1 WT Probe
42053 Fluorophore-2 ATA TGA TAG GGA GCT GGG GTG T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40546 0.40 uM
40547 0.40 uM
40548 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.