Protocol 43683: Probe Assay - Nudt16l1<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=  134 bp

Wild Type = 144 bp

 
>chr16:4938591+4938734 144bp CATCCCAACTCCTGTGATGTT CCACTGCCTATGAAATCATGC

Sequence

Large deletion:  Mutant sequence with junction in uppercase

catcccaactcctgtgatgtttactgaacattaatcaatgtgccagagaggggcaaagcataagtaaggcttaggtcccCTggcagcttgaattgagctcttgtggtgatggtggagctgaaagcacaggccta

 

This mutation is a 2443 bp deletion beginning at Chromosome 16 position 4,938,671 bp and ending after 4,941,113 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
63685 CAT CCC AAC TCC TGT GAT GTT Common A
63686 CCA CTG CCT ATG AAA TCA TGC Wild type Reverse A
63687 TAG GCC TGT GCT TTC AGC TC Mutant Reverse A
63688 Fluorophore-1 CCT GTC ACT TGA GCT GGA GTC Quencher-1 WT Probe
63689 Fluorophore-2 CCC TGG CAG CTT GAA TTG A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
63685 0.40 uM
63686 0.40 uM
63687 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.