Protocol 30995: Probe Assay - Larp1b<em1(IMPC)J> Probe-Alternate 1
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

>chr3:40774103+40774275 173bp ATGGTGTGCATGTGGAAATCT TTACTGTCAAACTAAAATGATTAGCTG

 

Mutant=  178 bp

Wild Type = 173 bp

Sequence

Wt Sequence: ttgcaggagttggttctctctaccacgTGtgatctggaggttatatgcagggtattagcttggtgacatgtcctttccctgctgagctatttcagtagtcctctgacacgatgggtcagctaatca

 

Mutant Sequence: ttgcaggagttggttctctctaccacgTAaggtatgaaaaaaaaaagactaaaacttctgtatattatttaaaataaataaaatgtgtcttatttgcaaatagatatttatattaaaatttgtaaacaagcagggtatggtacatgtc

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
33927 GAC ATG TAC CAT ACC CTG CTT G Mutant Reverse A
33928 Fluorophore-1 TGA TCT GGA GGT TAT ATG CAG G Quencher-1 WT Probe
35924 ATG GTG TGC ATG TGG AAA TCT Common A
35925 TTA CTG TCA AAC TAA AAT GAT TAG CTG Wild type Reverse A
35926 Fluorophore-2 ATG TGT CTT ATT TGC AAA TAG ATA TTT AT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
33927 0.40 uM
35924 0.40 uM
35925 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.