Protocol 33832: Probe Assay - Rnf40<em1(IMPC)J>Alternate1
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

>chr7:127589872+127589968 97bp CAGACCCAGGGACATCAGA CCCCACATCAAAGTCACCTC

Mut= 100 bp

Wt= 97 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case; and insertions with carrots ^g^ (CAT and CACATCA insertion):

 

TACACAAACCGGGTGGTGAGGGTGGGTGTGCCAACGGCGTGAATGTGATCAGAGAACAACTTTACGGAGTCCCTTCTGGCCTTTTACTTATGCGCACATATGTTCCCGTAAGTCCTGTGT^gatgtgtctgagttggaaacaggtaggggcaggaacggaatgtgaagctctcatgactagcatattctctttgcaagtgtctgagccttgctatatacctctcccctttcagtctaatccttgtcctattgtgtgtcagtgatggttcagggtcctgcccgacactctgcctcttcccttctggtaggaggagatggattccaaggtactgcagttcaagaacaagaagctggcggagcggttggagcagcggcaagcctgcgaggatgagctccgggagagaattgagaaattggagaagaggcaggccactgatgatgccacactcctcatcgtcaaccgctactgggcccaggtggatgcctgaagagcgagcgtcctactgggagaagccctgaggcattcctaacctcttgttctttcttctttcagctggatgaaactgtagaagccctcctccagtgctacgagaaccagagggaactgtcttcagggacagaggtgcctgggtgccaagagggcctgacccgtgatgtgatccctcgaccagacccagggacatcagatctgaggggtaggaccagggccctggg^GAGCTTATGTTCTCAGAAAGGTTTTGAGGAGGAGGTGACTTTGATGTGGGGCTACC^gaaatgcccggcacttggg^AAGCAGAAGTTGCTGTCCTGGAACTCACTTTGTAGACCAGGCTGGCTTCAAACTCAGAAATTTGCCTGCCTCTGCCTCCCGAGTGCTGGGATTAAAGGCGTGTGC

This mutation is a 593 bp deletion beginning at Chromosome 7 position 127,589,325 bp and ending after 127,589,917 bp (GRCm38/mm10). There is a 3 bp (TAC) retention 95 bp (7:127589325-127589419) after the beginning of the 593 bp deletion followed by 495 bp of additional deleted sequence. In addition, 56 bp after the deletion there is a 7 bp insertion (CACATCA) and a 19 bp deletion (GAAATGCCCGGCACTTGGG), that will not alter the results of the deletion.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40562 CAG ACC CAG GGA CAT CAG A Wild type Forward A
40563 CCC CAC ATC AAA GTC ACC TC Common A
40564 CCT TCT GGC CTT TTA CTT ATG C Mutant Forward A
40565 Fluorophore-1 TCT GAG GGG TAG GAC CAG G Quencher-1 WT Probe
42296 Fluorophore-2 AGT CCT GTG TTA CGA GCT TAT GTT C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40562 0.40 uM
40563 0.40 uM
40564 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.