Protocol 36859: Probe Assay - Actl6b<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

>chr5:137562951+137563119 169bp TGTGAAGTGTGTGTGTATAGGAATG CCCGAGTGTTCCACTGTGA

Mut = 190 bp

Wt = 169 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 CGTATTTGTTCTGAGTATGGACAAATGTGTGTGTGCACACACACATACATACTAACTGACATgtgggctgggcctccatctggactcttctcctgtgctcttctcacagtggaacactcgggccaagcgggagaagctgacggagctgatgttcgagcagtacaacattcctgccttcttcttatgcaagacggccgtgctcacagcgtatcctacatctcgggcagtcctgctgggtggaaggctagagtggggtgtgtgttgagctgggtggggctgggcctgattccctggagatcagtatctcgggtaggaagaatcctcttagcacctcactagtgcagaaggccccataGGCTGGAGGCTTGGGCTAGGGGCGGAGGTTGCAGATCTGTGGCCCTTCCCTATTTTACTTAGTTTATGGGTCAGTCTCAGCAGGCAGA

This mutation is a 293 bp deletion beginning at Chromosome 5 position 137,563,060 bp and ending after 137,563,352 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49911 TGT GAA GTG TGT GTG TAT AGG AAT G Common A
49912 CCC GAG TGT TCC ACT GTG A Wild type Reverse A
49913 GCT GAG ACT GAC CCA TAA ACT AAG Mutant Reverse A
49914 Fluorophore-1 CTG GGC CTC CAT CTG GAC TCT Quencher-1 WT Probe
49915 Fluorophore-2 CTG ACA TGG CTG GAG GCT TGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49911 0.40 uM
49912 0.40 uM
49913 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.