Protocol 44227: Probe Assay - Shkbp1<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=  111 bp

Wild Type = 100 bp

  
>chr7:27354755-27354854 100bp TTTCTCCTTGCAATGTGCTG TCATGGAGGAGGCTGGAA

Sequence

Large deletion:  Mutant sequence with junction in uppercase:

tttctccttgcaatgtgctgttcatgcctatgccctagAGgggatgggaattctgtacagagtcagtctttttgccttgtgagAAatgtGttagaccttcagaactctgca

 

This mutation is a 3202 bp deletion beginning at Chromosome 7 position 27,351,614 bp and ending after 27,354,815 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65048 TTT CTC CTT GCA ATG TGC TG Common A
65049 TCA TGG AGG AGG CTG GAA Wild type Reverse A
65050 TGC AGA GTT CTG AAG GTC TAA CA Mutant Reverse A
65051 Fluorophore-1 CCT ATT ACC CAG GGG TGT CCA T Quencher-1 WT Probe
65052 Fluorophore-2 ATG CCC TAG AGG GGA TGG GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65048 0.40 uM
65049 0.40 uM
65050 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.