Protocol 37650: Probe Assay - Fancf<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

>chr7:51861204-51861309 106bp GCCTTGGATGGAGATTTCG CAGCCTGGGAACTGAGAATCTA

Mut = 99 bp

Wt = 106 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GCTCATGTCCAATCGGCTCGCGCCTCGGGATGTCAGGGCGGAAGCGGCcgccatggagccgcttgtgcagcaaaccgagcgattctccgagctgttggccgtgtcgtgtgggtcgctggtgagcacgtgggacgccgaaaaggtgcgcagggcgctgcagtgggcgcgttatctgctccacgtgtaccgacgcttcgcgggccgcggccgcgtgcgggaggcgctcgagaggcggctccccgcgcggggagggccgctcgggctgcgcagcttcgcggcgctggagtccggggacgcgcggctcgccctccgcctgctgcggaaccgcgcgctcgcgcccgccgccgcccgcgcgctgccgagcttgctgttcccgggccccgccgcggaccaccgagacgacgtgccgcagtcgcgcctggtcctcctggcgcgccgcggaagcgcgctgcgcctgctgtgccgcctcggcggggacgcgcccaggagcgcgctgctgcgcacgcacgcggagctgctggacgcgcgcctgcacgagctgggcggggccgactccgcggccgcgcgcaaactcctggacacgttgtggacgcgcgggccgcgggagcacgtgctcgacgtgactgccgaagcgctgctgctacgcgaggaagacccggagccagcccaggccacggaccctgcgggtgcggatgagacacagaaactactgcgctggcttctcgaaagtcccgaggtattggccgctttttgccgccatctgccagccaagcgcctggcctccgtggcgggctgccaccacgcactgtctcgcgcctacctggacctgctcacaacgtgggccacgcggctgcactatgatctgcaaaagggtgcctgggtccctacacagatggaggacatgccctgggaagagctgtgcctaaggctgcagagcttgtgccatgcccagccatttctgcaggaagaggtgctggtgaccctaaggtcccgcaaagccttggatggagatttcgaggttcctgggatgagcatctggacagacctcctggtggtgcTTGAGTGTGGGATCGTGCTAGAGTAGATTCTCAGTTCCCAGGCTGGG

This mutation is a 1010 bp deletion beginning at Chromosome 7 position 51,861,249 bp and ending after 51,862,258 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51862 CAG CCT GGG AAC TGA GAA TCT A Common A
51863 GCC TTG GAT GGA GAT TTC G Wild type Forward A
51864 GCT CAC GCT CAT GTC CAA TC Mutant Forward A
51865 Fluorophore-1 CCT GGG ATG AGC ATC TGG A Quencher-1 WT Probe
51866 Fluorophore-2 AGG GCG GAA GCG GCT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51862 0.40 uM
51863 0.40 uM
51864 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.