Protocol 37132: Probe Assay - Ube2q2l<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

>chr6:136401853-136401985 133bp GCCTGGCTAAGAGACAACCTC ACTAGTGTGGAAGCGAAGTGG

Mut = 149 bp

Wt = 133 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

ATGAGAAAAATTAATTTCTTCTCATGGAGAAAATGAAGTCTCTAGCCTGGGTGGAGATCCCACATCAGTTGGGCTGGCCTATTGCCTGGCTAAGAGACAACCTCTTGGGAAGATGgattctccatgctaaaggggacagcaagaatatttattcctttctaagataatacatttttgatcaggcgtcttgccgggccacttcgcttccacactagtctcggaggagtggagaggaaggatgtcgtcaggtcttaaggctgagctggagttcctagcatccatcttccacaaggaccacgagcggctgcgcatcgtgagctggcacctggatgacctacagtgtcagttcctggtgccggaggcaactgtgggcagtccgccctcgccgccaccgctcacccttcactgcaccatcaccgagtcctacccatcttcaccggcgatgtggttcgtggactctgacgatccagatctcacatcgatcctggaacgcctggaaaattcgaagccagacagttcgcttagtcagcagctcaagtggctgatctgtgaactctgccgcttatataacctgcctgagcacctggatgtggagatgctggaccagccggtccccatcgctcagaaggccaccatcaaagatgggtcttcagaagaagacgaagatgcatcttacggactggacgactcggattattatgacatgaaagaagaagagacgagtagtgagaaaacgtcagagaatgaagggcttgagaaagaggatttggaaatattagagaatatccagaaaaacctttggcaaaagaatgcaagtggtgccacgtctggctcagtgtgtgcttcagatagacttatgaaagaactcagagaaatatacagatcgcagagttacaaatcagggactttttcagtggaactgataaatgacagcttatatgactggcacgttaaactgaggaaggttgaccctgatagttgcttgtacagagaccttcagcgcttgaaacaaaaagaaggcatagactatattttgcttaacttctctttcaaagataactttccgtttgatcctccatttgttcgagtcgtgttgcctgtactctctgatggttatgtcttggatgggggggcattgtgtatggaactgcttaccaatcagggctggagcagcgcctactccatagaatcggtcatcttgcagatcaatgccaccttagtcaaaggcaaggccagggtgcgctttggagtagataatcactatactgaacaagtggctagacgggtctataaatccatggtgctgaagcatgagaaatcaggctggttcactcctccacagaaagatgggtaaatagtagagcatcatgtatttggtatttgtagcagtagtgagaaagttattagctaattggtgctttagtggataatctctcaactgaccaccagtagaagtgtcagcctttccattttgcttactgtgggggtcttgggctgaactgtgagccttactgtggtttttcctgataaataatcataactccattttcagtcttccatgttccttgattaagtgaaatgtgttaagtaggccagtcagttgtgactttagataatgctggttattaggctactgggggggggataatgagtatttttaaacatttgttccatttagtactaataaaagtttttccagtgtccaatactgttttagaaaataaaataaaataaaataaaaaaagaataaaaaagaatacatttttttcttcccagaattcctgcccttgtgCAGTTGCTATGGTGTCAACTGTTAGGCCTGTCCATCTTTTTAAATCCTGACTCAGACAGGTACAACTTTATCTTTAAAACACAAAACAAAAAAATAACAAAACCCAACCAACTCCTTTTTTCTTTCTTTCTTTTTTTTTTAATCTGACAGACAGCAACTTTAT

This mutation is a 1677 bp deletion beginning at Chromosome 6 position 136,400,277 bp and ending after 136,401,953 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50611 GCC TGG CTA AGA GAC AAC CTC Common A
50612 ACT AGT GTG GAA GCG AAG TGG Wild type Reverse A
50613 AAG GAG TTG GTT GGG TTT TG Mutant Reverse A
50614 Fluorophore-1 CAT GCT AAA GGG GAC AGC AAG Quencher-1 WT Probe
50615 Fluorophore-2 AGA TGC AGT TGC TAT GGT GTC AAC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50611 0.40 uM
50612 0.40 uM
50613 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.