Protocol 31909: Probe Assay - Ube2i<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

Mutant=  80 bp

Wild Type = 110 bp

>chr17:25268957-25269066 110bp GATAAGGAGTGGCCTGTGGT TTTGTAGCCAGCAACCTTCA

Sequence

Wt Sequence (deleted region in lower case)

 

TCTCAGGGCTTTGTAGCTGTCCCAACAAAGAACCCTGATGGCACAATGAACCTGATGAACTGGGAGTGCGCTATCCCTGGAAAGAAGGGGGTAAGGGTGACTGCTGGCTTTGCACCTACTAACTTAACAGATCTGAAGGAGCAGAACTACTAGAAGGGACCTAAGTTTGCATATTTGTGACCACAGCTAGCCTGCCAGGTCACAGCTCTCCCCTGGATAAGGAGTGGCCTGTGGTGGTAAGGAGGCTTGgaccttggcaaatcactggatcagtgaacagcccggggtcaggggtaaagagcaggtgaaggttgctggctacaaagcagtaagccccaatggtgaggtgcccatggagaacccaggccgtgtttctttacactcatgggcctgagtgcagccggttgtctgcccttgtccagcacatgtaagagggatggtctcctataacttcaggcattcaccaaacccagacctctaggtcagcttagtgtctgagcaaaaacatagtttgggttcatctatacaaggctcagttgcagagtatgcagtaatggtggtgtagggcagttagagaagaagcccaaaggacccaggtttcaggaaacttctcctgggcttaccttggcaagtgagccactggtctaaccttggttccctccccacagactccatgggaaggaggcttgttcaagctacggatgcttttcaaagatgactatccgtcctcaccaccaaaatgtaagttgtgcaaagagcctgcagtccacttcccagcactaccaccaccctttgggagccatgtctgtgagtgcttggcctagtaggcaggtgccagtgctgggatagtcggtcctggtgagttgtcttggatgacaagtcccagggtagatgcgcatgtaattagtgtggtctctaatcccagcctattaggatgtaccacacatggcagagggagagttgtgtcaggctgaagcttctgggtgcttttgtgcccacaggagaactcgagtctgtaagttcccatagattacgtgagtggttagcatatatcagctgagttccattcagagcactaaaggagagaggagactcttactcatccccatctgctgtgttctgtgggatgctcatggccttcagaccatggggtcagaagagaaggctggactgatctctgttcagctcagcagaacttgcttgGGGGTACAGTGCCCACCACCACCATTCTGCCCAGGATAAGTAAGGCCAGGTCTGAGCTGGTTTATAAATAGATGTTACCTGATGCAGAGTTCCCCAGGAGTACTAAAACATAGTTTATTGGGAACCACTAGATGGAGTCTTGCCTGTTCTGTTTGGGATTTCCTTGTCTGCCTGAGTTCTTTTGTTAGTTTTTTCTTTTAAGGGGTATGTAGCTAGGATTGAGGTAGACAGAGTTGTTGGTCTCTTTCCTTTCGTGTGGTGCTGGA

954 bp deletion beginning at Chromosome 17 negative strand position 25,269,032 bp and ending after 25,268,079 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37953 GAT AAG GAG TGG CCT GTG GT Common A
37954 GCC TTA CTT ATC CTG GGC AGA Mutant Reverse A
37955 TTT GTA GCC AGC AAC CTT CA Wild type Reverse A
37956 Fluorophore-1 CAG TGC CCA CCA CCA CC Quencher-1 MUT Probe
37957 Fluorophore-2 TGA ACA GCC CGG GGT C Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37953 0.40 uM
37954 0.40 uM
37955 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.