Protocol 36767: Probe Assay - Rnf180<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

>chr13:105250481-105250588 108bp ATTTCCCTTTACTACCTGTAGGC GGAACATTTTGGAGTGCTGA

Mut = 105 bp

Wt = 108 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 CATTTGCCGTCCTGTCTGCTAATCAGAACACAGAAGGCTAGCCTTTTCAGTAGCAGAAATGAAATGTGCATGTGATCGGCTTTTTTGTTTTTTTTTTTTCTAAACCCATTCTCTGGTCACTTTACAATGACAGCATTGTCTTTATTTCCCTTTACTACCTGTAGGCCCagtggacagttgggaaactgaattgtcctttctgtggggcccgattagggggctttaattttgtcagcactccaaaatgttcctgtggccagcttgcagctgtacatctctgcaagagccggactgatcaccaagcagcacagggagggagactaatgagaccagcgctgaaacatttgccacatcccggagtcccgtcaggttgtgataaggaaactctgctgacaggtggtggctccaaaaccagaaaccactggcttttaagcatggcccgaaacagtaacggcctcggaagacttacagaagcactctgcctggaggtgcgagcgacgtattttgagatgaagaatgaaaaactgctcttcaaagcctcggacccaaaatgccagccttttgttcctcagcctgacaccggcaggtgtccttcaagagcgtctcacagaaagtcacacagtttggacctgaacatcagtgagaagctgattttattacccactttatatgaaatacatcgtaagcctactgcctatcccaggctaaatgaaacggggcccattgacctttcaggcttggctttaccgtgtagtaacagtagctgctcctttcaaagcccacctagttttgatccgaatatgctgctgcacagactttcagtggctccccatgagacccaggcacaaaggggaagagaatgccagtgcggcctagaagcttcttcagtctactcggatcacgctaacgctaacagcctgccattcctgatggatctgccctcagcagggaggagcgtgctggaggcctcggaccaagaggagcacctctcccagctggacttcctgcgctctgccagcttccccttgggcaccattaaccacaggctgaacaacagggagagaagcaagttgaggactctgcgaaggcagcagaggcgcgaacgatggctacagaagcagGTAAGCGCTTTGCAACCAGTTTAGCAAGAAATAGTGTCCCTAAGTACTGGGGTCTGTGGCACAGAGGTCAATGTGAATGTACGCATTCTGAAATCTTAGTCTTAAGTTATGAGATGATGTGTCAGTCTGTGT

This mutation is a 953 bp deletion beginning at Chromosome 13 position 105,249,611 bp and ending after 105,250,563 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49607 ATT TCC CTT TAC TAC CTG TAG GC Common A
49608 GGA ACA TTT TGG AGT GCT GA Wild type Reverse A
49609 CAT TCA CAT TGA CCT CTG TGC Mutant Reverse A
49610 Fluorophore-1 CTG TGG GGC CCG ATT AGG Quencher-1 WT Probe
49611 Fluorophore-2 CGC TTT GCA ACC AGT TTA GCA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49607 0.40 uM
49608 0.40 uM
49609 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.