Protocol 33048: Probe Assay - Nup153<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

>chr13:46717404-46717513 110bp AAGATCCAGAAGGATGAAAACA CACAACTGTTTCCAGCAAAATTAC

Mut= 95 bp

Wt= 110 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

CTCCACCAGCTGCACTAACCATATTTCTAGCTCTTTGTCTTCCTTAAAGAATGTGACACAAAATAATTGTTGAAATGACTTACAGTGTGTCTTTTAATGTATTAAGATCCAGAAGGATGAAAACATGTACTACCTACCACtaaatctagatcttggtataatgacataccattcacaaactgaatcttagtaattttgctggaaacagttgtgatttgcttggcatgactttctgtaagtacttacaaagataagttttctaaagtagtcttaatgtatgttttctccaacagggcattcttagcagggttacagaatctgttaagaatattgtaccagggtggctacaaagatacttcaacaagagtgaaaatgcttgcagctgttcaccagatgcagatgaagttccaccctggccagaaaatagagaagatgagcatgcgatttatgcggatgagaacacaaacactgatgatgggagaatcacccctgaccctgcaggcagtaacacagaaggtaaatactgtggatatttggttacaaagcgtagatcttcctaatttaaatatctttatattacctgagtgTGATAGCATCTGACCAAGTAATATATAAATCCTGTATGGAACATTTGGAATGAAGTTCACACTGAGTATGTTTATTGAGTAGTTTTTAAAAATATTTGTGTGTATATGCACATGGGCTGGCATGCCACAGTGTGTTGTGTGGAGATAAAGATCAA

This mutation is a 447 bp deletion beginning at Chromosome 13 position 46,717,030 bp and ending after 46,717,476 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40541 AAG ATC CAG AAG GAT GAA AAC A Common A
40542 CAC AAC TGT TTC CAG CAA AAT TAC Wild type Reverse A
40543 GAA CTT CAT TCC AAA TGT TCC A Mutant Reverse A
40544 Fluorophore-1 TAG ATC TTG GTA TAA TGA CAT ACC ATT CA Quencher-1 WT Probe
40545 Fluorophore-2 ACC TAC CAC TGA TAG CAT CTG ACC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40541 0.40 uM
40542 0.40 uM
40543 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.