Protocol 33840: Probe Assay - Rab28<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

>chr5:41703683-41703822 140bp TAATTTCCATCAAGGAGAAGGAG TCAGTCACGAGGTTGGAGAC

Mut= 148 bp

Wt= 140 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

AATTGAGATCTTAATTATATTGTTGCTTTAGTTCCCATTTACAATTCTCAGAGTCTAATTTCCATCAAGGAGAAGGAGCTTGAGAATCAGAATGCCTAATGAAGTCTGATTAGATTAGGCTACTGTTTCCTCTGctggttattgcaagtgcagtcagtattcctggggagggtgagtctccaacctcgtgactgattagagctgcaaaaagttgtttgttgagttcattaaagtatacatgttagtattacttaataagtttagaggtgttcttatgtttacattttttaaacaaagaaattaattttgctgttttgtgcatccattttttttttttttttagacctccttagctacgtgttttgctcaagagacatttgggaagcagtacaagcagacgataggactggatttctttttgagaaggattaccttaccaggtgagagactataaacctatgagactgtgtaggtctcgctcacagggcagccttactaggtaacagactatagaaccttacctgtgTGAGGTTATATAAGTCTCACAGGAATATTACCTGAAATTATCTATCATTAGCAACTAGTGTGAGCGTATACTGTCCACTGGGGTAAGAAAATGTTTATACAGTTCCCTATTCTATTGCTGTTAATAGCAACTCTGCAC

This mutation is a 392 bp deletion beginning at Chromosome 5 position 41,703,352 bp and ending after 41,703,743 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42311 TAA TTT CCA TCA AGG AGA AGG AG Common A
42312 TCA GTC ACG AGG TTG GAG AC Wild type Reverse A
42313 ATA CGC TCA CAC TAG TTG CTA ATG A Mutant Reverse A
42314 Fluorophore-1 TCA GTA TTC CTG GGG AGG GT Quencher-1 WT Probe
42315 Fluorophore-2 CCT CTG TGA GGT TAT ATA AGT CTC ACA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42311 0.40 uM
42312 0.40 uM
42313 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.