Protocol 37382: Probe Assay - Rfc5<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

>chr5:117385436-117385539 104bp TGACACTTCCAGCCACTAGC ATATCGATCCCTCGGTCGTC

Mut = 97 bp

Wt = 104 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

AATGGCTGGCTGACACTTCCAGCCACTAGCTTGTAGCTGGCAcagggtgtgactgtcacagcatcctctcttcctcctgtagctgaatgcttctgacgaccgagggatcgatattgttcgggggccaatcctcagctttgccagcacaaggacaatcttcaagtaagaattacctgtaggatttctctccctacaggtcttagtgcgggttaGGAGGGACACAGCCTGGCTGTTCGGTCCCCCTTGGCTGGTATAAAGGGAAGAAGGGAACACCTGGACAGAAGCAG

This mutation is a 170 bp deletion beginning at Chromosome 5 position 117,385,338 bp and ending after 117,385,507 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51198 TGA CAC TTC CAG CCA CTA GC Common A
51199 ATA TCG ATC CCT CGG TCG TC Wild type Reverse A
51200 CCA GGT GTT CCC TTC TTC C Mutant Reverse A
51201 Fluorophore-1 CTG TCA CAG CAT CCT CTC TTC CT Quencher-1 WT Probe
51202 Fluorophore-2 AGG AGG GAC ACA GCC TGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51198 0.40 uM
51199 0.40 uM
51200 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.