Protocol 48109: Probe Assay - Rbm28<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

Mutant=  103 bp

Wild Type = 109bp

>chr6:29158547-29158655 109bp CCGTAGTCCTGCTTGATAAA GCAAAGATCATATTGTCTAGGG

Sequence

Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion):

Large deletion:  Mutant sequence with junction in uppercase

 

This mutation is a 447 bp deletion of Chr6:29,158,595-29,159,041 (GRCm38/mm10)

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
72618 GCA AAG ATC ATA TTG TCT AGG G Common A
72619 CCG TAG TCC TGC TTG ATA AA Wild type Reverse A
72620 AGA TAC AAA GTA AGC TGT CAT C Mutant Reverse A
72621 Fluorophore-1 TGG GAA AAG TCA CAT TAA TTT GT Quencher-1 WT Probe
72622 Fluorophore-2 TCA CGA TTA GAA CAG CAA ACA ACT G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
72618 0.40 uM
72619 0.40 uM
72620 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.