Protocol 36632: Probe Assay - Noc4l<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

>chr5:110652473-110652581 109bp GGCTCAGAAATCGTCTTTCC CTGACCAAGGTCCCAAACTC

Mut = 101 bp

Wt = 109 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 ATAGACCATGCGATCTGGGAGGACGACGCAGTCACTCATTGTAGCTCAGGAAGCGAGTTGAGATCCGTATACTTGCCAGGACTGCGTGAATCCGTATACGTAGCCGTTTTCACTCACTCACACACACACACACACTCTCTCTCTCTCTCTCTCTCTCTCTCTCACACACACACACACACACACACACACACACACACACACACACACACACACACACCCATGTTTTATTTGCGCCATTTCCGAGTTTGATACTGTCTCTGTCCTAGTGGGGAGACCCAGGTTCTGAGGCAAAGCGAGAGGAACTACATTTCAATACCCCCTTCCCAGTTTGCAGGGCTCAGAAATCGTCTTTCCCAAGCCCCGGtctggcatcttaaatagtttacagctgtgaagatgagaaacagccggaagattggggctgagtttgggaccttggtcaggcttggcacacataaatatgagtgtgagttagctctcctaactctgtccccagggtcccagggagccacatacaaatacaaggtgtggataagacatcgataccacagctgctgtaaccgcctagaggaactcctggcccatcccaccttccaggtcaaggtgagtcttgaagcccttagcccagcttttcctctcatctctaaAGTACTTCACAGCACAGGGGTAGAGGCCTTTGATGCGAGCATCTCTTCCTTCACTTGTCTGAGAGACTTCTACCTAGACATCTTTATTGCTGTTCTTTTCCACTGTGTGCTGTGAATCATCCGGACCTAAAGACATGGAGTCTGTAACCCTAGTTCCAGGCAGCAGATCTGCTTTGTACATTGTCACATTTGTTGAGGCACCTCCAAAACTGGGGGGGACAAGGGAATAGTTAGGTGGCCTCCCCTGCAGGCCCACCCTGTGTACATGCCTCAAGCCAAGTTCTGTTAAGAGTTCTATCCCAGGTCACCATCGGCACACACATGTAACATGGA

This mutation is a 283 bp deletion beginning at Chromosome 5 position 110,652,269 bp and ending after 110,652,551 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49260 GGC TCA GAA ATC GTC TTT CC Common A
49261 CTG ACC AAG GTC CCA AAC TC Wild type Reverse A Wt R
49262 AGA AGT CTC TCA GAC AAG TGA AGG Mutant Reverse A Mut R
49263 Fluorophore-1 AAC AGC CGG AAG ATT GGG G Quencher-1 WT Probe
49264 Fluorophore-2 CCG GAG TAC TTC ACA GCA CAG G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49260 0.40 uM
49261 0.40 uM
49262 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.