Protocol 35757: Probe Assay - Rsu1<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

>chr2:13224647-13224768 122bp CTCTGTACCTCAGAAACAAG AGGGGCTGAAAGCAGAAATG

Mut= 121 bp

Wt= 122 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 

GAGTTCAATGATAACAAGGACAGCTCCACAGGTACGGGATCATTTAAGAACTTGAGTGTACCCTCCTTCATTGGGGTTAGCTGTCTTCGGCTGGCAGTTCAAAGCAACCACAAGGAAGAAAATAACTTCAGCTGTTATGCTTGTAGTGTGGAAGTGTGTGCTGTGTTCTATTCCCTGAGAGGCTGCTGACAGCATGGGTCTCATGTCTTCCTCATTTCTGCTTTCCGCCCCTCTGTACCTCAGAAACAAGTCCTGCCAGGTAGTTTGATCTAATCAGACAAAAGGAAacacttcctgagaggctgctgacagcatgggtctcatgtcttcctcatttctgctttcagcccctctgtaactcagaaacaagtcctgccaggtagtttgatctaatcagacaagaggaaacacttccgttccacctggtcaagaatttttagccatgaaagtaagaaaacccacaaaatgcatgtggacagcactggggtttgaccttacttgcgttcaatagggaagtgtgtggctagcatcctttgccccttgaattcacatgctgcatctccatccctatagctgtgccaccaaatgtagcggaactgaagaacttggaggtactaaacttcttcaacaatcagatcgaggaactgcctacccagatcagcagcctccagaaactcaaacacctgaaccttgggtgagtattggaggagcttgaggaaatctccgccaggtaatgagctgaagccaatgcatgctgtgggcaagcaaGGGGAAGCGCTTATATGATTATCACATGGGAGTAGTGGGTTTCTAATTGCTTTTAATGTGTCAGGTTTAAGAAAAGGCTTATTGTTTTAATATTTCCTTTCAGTCTTCTCCATTAGTGTATATCCAGTTAAAGCTAATTGAGAAGCTAATTGTTGGCACAACAAGGAATCTTTTCATGATATTTTGT

This mutation is a 481 bp deletion beginning at Chromosome 2 position 13,224,231 bp and ending after 13,224,711 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46951 CTC TGT ACC TCA GAA ACA AG Common A
46952 AGG GGC TGA AAG CAG AAA TG Wild type Reverse A
46953 CTG ACA CAT TAA AAG CAA TTA GAA ACC Mutant Reverse A
46955 Fluorophore-1 ACT TCC TGA GAG GCT GCT GA Quencher-1 WT Probe
46956 Fluorophore-2 CAA AAG GAA GGG GAA GCG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46951 0.40 uM
46952 0.40 uM
46953 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.