Protocol 33082: Probe Assay - Rnf214<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

>chr9:45900465-45900569 105bp GCACTTAGTATAGGACACACTCAAAAC TTCTCCACCATCAGAACAAACTTCT

Mut= 90 bp

Wt= 105 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 

ATCTAAATATTTATGAAAATATGCATAACTAAGTTAGTATATGTAACTACCGTAACTAACATTGTGCTATTGTTTCCTATTCACAAAAGCTGTTGGCAGTGGGCATTTATTTTTAGATCTTGAGGGTCACTTGTTTTTAAGCACTTAGTATAGGACACACTCAAAACTTTTGCAAGGTCTCACTGACCAGGTTAgaatacctacaaattgttgggtagaaagaagtttgttctgatggtggagaaggcctatagcatctaggtttgttctagtggctgtgttggggatgtggctcactagtagagcactgtccagcatgggtttgatccccagcactggtgctagtagcagaatcctgggtctttggtgtgctagtcaggtgacataccactgagctacaacctcatcccagattttttttttttttttttttttttttttttttttggatagccccaaagactctgcccagaaacagaagaacttgtctccgccgagtgtaagtagccaaatgataaccaaggagagtaacagaaatgcccatttggaacatccagaacagaatcctggttcatcagtgggtgatacttcagcagcgcacgaggaagttgtaggagaaaacttggtagccacagccctttgtctttctggcaatgggtctcagtctgatttgaaggacttgaccaacccagcaggagaggagggggacacgagcctccgggagagcctccatccagtcactcggtctcttaaggcagggtgccactcaaaacaacttgcctccgggaattgctctgaagagaaatgcccagcagcctccgtcctaaaggaaggtagcagggatgcaggcttggacttgctacctgtagtaccgcctgcaaatggggttgaaggagtgagagtggatcaggatgatgatcaggatagctcttccttgaagctttctcagaacattgctgtgcaggtcaagtatcacatttctactactaaaatggaattggggacgggaggggagaagccatttaggtggtagagaactgcattgtgtcattttttgctagtagtcatgacacagtttgcatatagtatcatgtaatggcttcattctgctattgcagaaacaagaaagccagtatctaattgtataatatgcaggaattattatgacctatgtgtaacctctttgGATGAAAACTTGATTGGTGTAAATCCTTCCTGTTCTTTCCTTTTATTTTTTTCTTTTTCTTTTCCTTTTCCTTTTTCCTTTTTCCTTTTTCCTTTTTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTCCTCTTCTCTTCTCT

This mutation is a 992 bp deletion beginning at Chromosome 9 position 45,899,524 bp and ending after 45,900,515 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40632 GCA CTT AGT ATA GGA CAC ACT CAA AAC Common A
40633 TTC TCC ACC ATC AGA ACA AAC TTC T Wild type Reverse A
40634 AGA ACA GGA AGG ATT TAC ACC A Mutant Reverse A
40635 Fluorophore-1 AAT ACC TAC AAA TTG TTG GGT AGA A Quencher-1 WT Probe
40636 Fluorophore-2 CAC TGA CCA GGT TAG ATG AAA ACT T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40632 0.40 uM
40633 0.40 uM
40634 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.