Protocol 34377: Probe Assay - Nup205<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

>chr6:35186591+35186725 135bp GCTTCTCCTTGCTGGTAAGTTG GCAATGCGAACTCAGGTATG

Mut= 141 bp

Wt= 135 bp

Fam=Mut

Hex=Wt

HOM Lethal (DJL 7-29-19)

Sequence

Wt Sequence (deletion in lower case):

 AAGGAGGTTTTAAGGGGCCTAATGAGGTGGCTTAGTACTAGAACACTTGCCTAGTGTGTGTCCATTTGTGGGTTACATCCCTACCACCACACATATAAAAAGAGTTTTGTAGGATATAAAATCATGTTACTAATTTATGATTCACTCTACaaagtaatactattgcttgttacttaatatagttcatatgtccatttgaattattgtatgtaactagaacattcacttagatatgtggaaatcagaatagtatttgtatttttcctggtttttatggagcttagcagatagttttgagaaggtagtactgggaaagatgaaggtggtgccacgtgttgcctcatatcccctagttctggataactagataagaaacacttcagtttgtcatgctaccaggcttcattgtttttgttcgcatgcatgctggccatgactctacttgcctggcctccaaatataggattaaaggcatgtgctgccttggcaaacgaggctgcgttgtcatgaactgacttctgctaggagcactattttcattacttaattataattttatcattctagcccaaaaatgttcaacagcatgagaaaattcagaaagccagttcagagggtgttgcgatccaggggcaacaaggaaccagacttcttcctgagcagctcattaaagaagcatttatcatcagtgacctttttgatattggggaattgtcagctgttgagcttctccttgctggtaagttgacatttagctggaagtgtgatatgtagtcagaatcatgaagctagaacccctaTCTTGGACTTTGGTTTTCTTTTAAAGATTTATTTATTTCGCATACCTGAGTTCGCATTGCTGTCCTCAGACACACCAGAAGAGGGCACTGGATCCCATTAGAGATGGTTGTGAGCTGGGATTTGAACTCAGGACCTCTGGAAGAACAGTTAGTGCTCTTAACCACTGAGCCATCTC

This mutation is a 605 bp deletion beginning at Chromosome 6 position 35,186,016 bp and ending after 35,186,665 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38394 GGG TTA CAT CCC TAC CAC CA Mutant Forward A
43456 GCT TCT CCT TGC TGG TAA GTT G Wild type Forward A
43457 GCA ATG CGA ACT CAG GTA TG Common A
43458 Fluorophore-1 AGC TGG AAG TGT GAT ATG TAG TCA GA Quencher-1 WT Probe
43459 Fluorophore-2 ATG ATT CAC TCT ACT CTT GGA CTT TG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38394 0.40 uM
43456 0.40 uM
43457 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.