Protocol 34918: Probe Assay - Nap1l5<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

>chr6:58907072-58907168 97bp AGTGAGCAGCCATTCACTTG GGTCCAGAGTGCGTCTGAAG

Mutant=  94 bp

Wild Type = 97 bp

Sequence

GGGGGGGGGTGTATAAACAGGTATGGAAAAATACAGTCTCAGCCTCGAGCTGGTTCCAGCCAGTTCTGCAGGGGTGGGGGCGGATGCTGAGGGCTGGGTGTCTAAGTGGCAGGTTTTAGAGACCTCGGAGATAGCAGCATAGCAACAATTTGGTGCAGTGTGTGCGCACAACCATCAGCTGATAGTGAGCAGCCATTCACTTGCCGCGAGCcgcgaagcggccgcgcggacgtcaccacagccgtcagcgtttgccgcatcttcagacgcactctggaccagccgcaatcacagctgcaggacctctctggaccagctctattgcagactgcgcaaccaccagaccactgcggcaaacaagcccagctgagccgagcagtagccatggccgaccccgagaagcagggacccgctgagagccgcgccgaggacgaggtaatggaaggcgcccagggtggcgaagatgccgcgaccggtgacagtgccgctgctcccgcggccgaggagccccaggcccccgcggagaacgcgcccaagcccaaaaaagactttatggagagcttgcccaattccgtgaaatgccgggttctggcgctcaaaaagctgcagaagcgctgcgataagatcgaggccaagtttgacaaggaattccaggctctggagaagaagtacaacgatatctacaagcccctgctcgccaagatccaggagctcaccggagagatggagggctgcgcgtggaccctggagggagaggatgatgaagatgacgaggaagaggacgacgaggaggaggaggaggaagaagaggctgcagctggcgcaactgggggtcccaacttcgccaagaagtgagcacagcaagctgacaggtgataaatcccctgccccttttatttccaatgtcggcagactttaaaaggctcaggatttgcccaaaataaaatgtgaatctattctgacattcctaaaattaaattgaatcaagtagattctggctagcccgtttctaatctgacttttatttgtgtaaaacagatgctaaaagacgttttttagaaaaaaaaaaaagcctaattaaacttttttttttctccctgctcttctgtgagaactggacttgagactgttgaaaggttgtcagttagatgggaattaaaagtgcgtcacacgttgaaatccattcatcacactacaccagaacatccaagctaagacagaactcttctcaatgcttaattcttcagtttctttacattttccagcgcagaggaaaaggaacccaggaacgacgtcatctttaagacttttgcttttgcaaacccagacatcagctttacactccagaggagccttggcatggagcaagcctggactgacagcatttactgattatgtggctaggaaaactgccatttcaagttgtgaaaaaatgttttaaatatttgcatttacagaaagaacatggtttcaaaaatgagggtgtattccatgtataatattgtcaacacgtgttcatctgttatggtcccgtgttacctgttttcttggtagtgtttgtttacaaaatcgtaaaaaattaccccaaatgttttaaagtgttaaattaccttatagcattttagaaatataatttacttgaagagatgtagaatgtagcaattctgtaaagcatgtgtatccagtgttgcctagtttgacctttgtgaagtctttttgtcttgtagcttttagcaagtagctgtgaaaaccatcagaactcctcaatgaagctaatgtttggaaaaagtatatacttgaagaacaaacccaagtgtgtacccccaaccccagctcagaaatagaaagggtttaagtttgcttgtattagctgtgccttcattattttgctatgtaaatgtgacttaataaatggtgcaaaattaaattttattgcttgaggacagacgggcataaaggaagggaaaagacatttaatgtaaaggcctctagcttctttgtgggttttgagttgtctgtgaaccaatgttctgtaaagaaacacaaacttaaagttgtttaccactgtggtgttaacaaaacagtattttcaaaaataaaaaaatccttgttattctgactacctgtactgtcatctataaaagtgggtgggaacaatcccttcctcgcagttattatgaagagaaagtgagtaactacgcttacagcccacacagcagcgtctgacacctaggaataatttccagctattattgttttaataaagacaaaagacccagatagaaaattcaactccctcccctgctacacaacagaaaatggtagcattattaatttctaacttggtgattccttgttatcagaaaagaatatggcTTGTAATTTAGAAATATAGGACATTCAAAAACTATCTTGTTTTAGTTTCCTCCATATAAAGCCAAGTTTTAATTTCCTCTTAGACAAGGTAAGATTTTAATGTGACAATATGAATGCACACACACACATGCACAAACAAACCAAAAAATAATCAGACGCCATTCTTTTCCATTTGCTGCAAGGGTTAATCACACTGCAGCAAAATCT

 

A 2165 bp deletion beginning at Chromosome 6 position 58,904,976 bp and ending after 58,907,140 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44798 AGT GAG CAG CCA TTC ACT TG Common A
44799 GGT CCA GAG TGC GTC TGA AG Wild type Reverse A
44800 CTT GGC TTT ATA TGG AGG AAA C Mutant Reverse A
44801 Fluorophore-1 CGC GAG CTT GTA ATT TAG AAA TAT AGG Quencher-1 MUT Probe
44802 Fluorophore-2 TCA CCA CAG CCG TCA GC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44798 0.40 uM
44799 0.40 uM
44800 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.