Protocol 36317: Probe Assay - Nsl1<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

>chr1:191070326+191070441 116bp CCAATGAAGCAGGTCTGG AGGCAAAGACAATGGCACA

Mut= 121 bp

Wt= 116 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

 TTATTTTTTTTTTAAACTGCATCTGTCTGCAGTTACTTCTTCCAGGGTGCTTCATTTCTGTGCTCTGTCCTAGACCTTCTGAGTCCCGATTTACACACTAGTAAGGTTCCTGGGCAATCACTCCTGCTCGAGCTTGAGAACCACTGTCTACAGCCGAGGAAAGAGATGATAAAATAGTTGCCACTGAAGTCTGAGGAAGTCCCCCAAGTCACCATGCCTTTCCTTTCCCTGCCCTGGTGAGTCAGGTCCTAGAGTGGGTGCCCTTCTCACTGCTGTCACAGGTTGGGTTGCACAAAAATAAAGTGAACCCTGTCCATGGTATGAGATGCCTTTTATAGTTTAATTATGGTGGGGAAGGGCAGAGGACTGGCCCAGGGCCCTGCCCATGCGAGGCCCATGCTCTGGCACTGACTGCATCTCTGCCCTCCTCTCTGTCACCTGTCCTATGCTGTTTGGGTCGATCCGTTTGCAAGGCTCAGTGCCGTCAGTTTCCTCTCCCCGAGGTCCTTCTCCCTGAGCGATCTCATCCAGTGCTTTGTCCGCACCTGGATTTCAAGCCTAAGTGCTGTTCTTTATCTCCCACTGCTCCTGCTGCCGTCTTCTGCAGCCTTCGTCTCTGTGGCCTGAGGCACTTATCAGGTGCCTCTTTCTTGGGACTATGCTGCTCGCCTCGTGTTTCCTGGCGCTTGCAGATTAATCAGTGCGgagatgaggggggctccgttaacctgccatgtttgttttctttattctgtgacttatgcttatataatatttctatactaataatggacttctctgaaatagattctgacatcaaagtgcttgaagatgaatttgatgagctcatagtagatgtggcaaccaaacggaggcagtatcccagaaggatccttgagagtgtcatcaaaaccttgaaagcacagcacgcgagtctggtgagacgcttatatctaaatccctacacagtattttaccacagtagagagttagtttacttacctttcatatagtgcaggctggtcttgaacacctgatcttccttctctcactggctggggtcagtcaccatacctatcacatatcagtcttctcagaattattgaaactggagtaaatgcaaatgagccctgaatcataatgccaatgaagcaggtctggcagcatagacctgaggcaaatgctgagGAGATTGGCTGGGTGCCATTGTCCCGAGAAGTGACCACAGCAGGCTTTAGATTGTGCCATTGTCTTTGCCTTTAGTTTTTTAAGTCAGCAGACTTTTAAGTGTGACCTTCCAGTATTGTCTCCTTCAGTAATGCTTTGGGGCATTAAAGGCCTGCACTTGACCTTAGAGTCCTGCTGTGACAGGAAAAAGAAGTCCTTGAGGATGTTCACTTTCTCACACATTCGGCCTTAAAGAGCCCTTCCCACACTGCAGAGCAGAGAAAGCGTTATCTGCCGTTGACTGTGTCATGCCTCCCTCTAACTTACCTCCTTGACCTGCTAGAGAAACTCTGGAACTAGTCAGCTCGAGGAGGGGGGACAGGCCTGGAGAGCATGAGTGTGCCCTGTCACTGACTGTGGCAGGTCCAGCTGCTCCTGTGTGCTTCCGTGAGGAAGACGGAAGACTGCTGTGGTAGTGTAGCCACTTTTTTGAAGTTTCTGTGGAAATAGGAGTTGTGAGTTCCAGATATTTGTCCTGAATGAGCATCCGCCACACTGCGGTCCTCGCCCGGTATTTTAGAGCTGCCTTCCCTGTTTCCTCTGACTGCGATCTTCATAGTGCTGTTTGTTACTCTGCAGCAGAGGGCGGTGCATAAAAGGTATTCAAGCCTAAAGACATTTGCCACCCAGAGTTCAGGAGCTAAAAAGGGTTAAACCTGGAGGCCAGCACTTTTGACTGTGTGTCTGCTCATACCAGCTGAAAGAGAGGCTGAGTCATCTGAGGCATTACGTGTCTAAGGTAAATGTTTTAATTTGTCCTGTGTTTAGAAGCAGTATCACCCTGTTGTACATCCTCTGGACCTGAAATGTGACCCTGACCCAGGTGAGCCAGTGTGA

This mutation is a 483 bp deletion beginning at Chromosome 1 position 191,069,888 bp and ending after 191,070,370 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48414 AGG CAA AGA CAA TGG CAC A Common A
48415 CCA ATG AAG CAG GTC TGG Wild type Forward A
48416 ACT ATG CTG CTC GCC TCG T Mutant Forward A
48417 Fluorophore-1 AGA CCT GAG GCA AAT GCT GA Quencher-1 WT Probe
48418 Fluorophore-2 ATC AGT GCG GAG ATT GGC T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48414 0.40 uM
48415 0.40 uM
48416 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.