Protocol 45309: Probe Assay - Cfap20dc<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

>chr14:8644673-8644772 100bp CGTGCACATGGTCTATGCTC ACTTGGGGCACTGGTGAAAT

Mutant=  118 bp

Wild Type = 100 bp

Sequence

Wt Sequence cgtgcacatggtctatgctcacattctcttatggcatagtgaaagaGtgaataaaaatcctgtttttgattttcataaaaatttcaccagtgccccaagt

Mut Sequence: cgtgcacatggtctatgctcacattctcttatggcatagtgaaagaGTgacttaccaggttgacatgaagtaattaagataatttaattgtatttcttggatatcactgtacgtgtca

A 442 bp deletion beginning at Chromosome 14 position 8,644,284 bp and ending after 8,644,725 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65262 CGT GCA CAT GGT CTA TGC TC Common A
65263 ACT TGG GGC ACT GGT GAA AT Wild type Reverse A
65264 TGA CAC GTA CAG TGA TAT CCA AGA Mutant Reverse A
65265 Fluorophore-1 AGT GAA TAA AAA TCC TGT TTT TGA TTT T Quencher-1 WT Probe
65266 Fluorophore-2 TGA AAG AGT GAC TTA CCA GGT TGA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65262 0.40 uM
65263 0.40 uM
65264 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.