Protocol 34651: Probe Assay - Nfyc<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

>chr4:120790560-120790661 102bp GCTTCAAATGTCTTCTGTGAGG TCTTTTCTCAGCTGTTTTCACTGG

Mut= 109 bp

Wt= 102 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AGATCCACCTACCTCTGCCTCCCTGCTGGGATTAAAGGTATACATGGCCACTGCCTGGCTCAATACTTGATTTTCTAAATTGGAACTTTTTGATAGTCTGTGATGTAATCTAGGAAAATTTTGCTTCAAATGTCTTCTGTGAGGATGTAATAAAGCCTATCAGCACagcacagaccttaacactgaatgaaacaattaaaccagtgaaaacagctgagaaaagataagtaactagcctggtgagaaagtatgactttattatttctaatgtgctttgtgtctgtgtgttttcagttgtgaaaatgtccacagaaggagggtttggcggtaccagcagcagtgatgcccagcaaagcctccagtccttctggcccagagtcatggaagaaatccgaaacttaacagtggtaaggaaaaacaaggagcttctaaagcagatcagtagaaacaagactcggGCACTTTTATTTTCAAGCATATTCAGGCGTAGTGTATTCCTGACCTGAATGTTGGGAAATAAAAATAGAATTTAACTTGTAGGAGGCAGTACCAAGGTGTGTTTGTGACAGTGGCCATCTGTTTAGATGTTCTGCTTGGAGGCAGGCTCATGTGGAACTGTGCTTTAGCCTATAGA

This mutation is a 292 bp deletion beginning at Chromosome 4 position 120,790,326 bp and ending after 120,790,617 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44147 GCT TCA AAT GTC TTC TGT GAG G Common A
44148 TCT TTT CTC AGC TGT TTT CAC TGG Wild type Reverse A
44149 TTT CCC AAC ATT CAG GTC AG Mutant Reverse A
44150 Fluorophore-1 AGC ACA GAC CTT AAC ACT GAA TGA Quencher-1 WT Probe
44151 Fluorophore-2 CAG CAC TAT CAG TGC ACT TTT ATT TT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44147 0.40 uM
44148 0.40 uM
44149 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.