Protocol 36641: Probe Assay - U2surp<em1(IMPC)J>Alternate2
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

>chr9:95493701-95493827 127bp ACGTATTTAGGAGGATGAGAAGG ACCTTTAGCTGCATTAACAACC

 Mut= 148 bp

Wt= 127 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AGGTTTGTGTATTTTGTTTCTTTTTCTTTCTGTATATGGGTGTTTTTGTCTGCTTGAATGTGTGTGCCTGGTATCCAGAGAGGTCAGAAGAGGGGGTTGGTCCCTTAGGACTGGAATTAGAGGTGGTTGTGAGCTGCAGTGTGCGTGCTGGGTCTTGGGTTCTTAGGGAGAAGAGCCAGTGCTCATTACTGGTGAGCTTCCACTCTCTGTGTTGGCATTTGCCTTTAAGACAGTATTTGGCAGTCAGATCTCTGTTCAAGGCCAGCCTGTCTGGATACTGAGTTCAAGTCTGCCTGGCCTCCACAGTGAAATCTTCTCTCAAAACAAAGCAAACTGAATCAGTCAAACCATACCCAAACAgcagttcttgtgtaactaatatttcgttacgtatttaggaggatgagaaggcagcggccgaaatttatgaggaatttcttgctgcttttgaaggaagtgatggtaataaagtgaaaacgtttgttcgaggcggggttgttaatgcagctaaaggtaagtaccaatatctggctttgaaaacaacccttgGATAATGTGATAACAGATTAATTATGGAAGACATTTGACTTTGAATTATCACTTTTCTTTTCTATTAGCACCCTCTAGTGGTTAGATTAATAAACAATATAAATGATCAAATATGAAGTAATTAAGACTCAAATTTGATTAGTGGGACTGTATGAAATAGATAATAATAATAGTAATAATGATGATGATAATAATAATAACTAGTACCTTTCAAGTTTAAAAAAAGTGAATTATGTTACAGAATTTTTAATGTTTTATAGTTTTGCTATAAAGTGGTCAAATTGTGATTTTATTTTTTCAGGTTGAGTGGTAGTATGTAAATTTAATTTTATATTCAAATCTTAATACTAAAATA

This mutation is a 189 bp deletion beginning at Chromosome 9 position 95,493,667 bp and ending after 95,493,855 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47650 CCA CAG TGA AAT CTT CTC TCA Mutant Forward A
47652 AAT CTA ACC ACT AGA GGG TGC T Mutant Reverse A
49283 ACG TAT TTA GGA GGA TGA GAA GG Wild type Forward A
49284 ACC TTT AGC TGC ATT AAC AAC C Wild type Reverse A
49285 Fluorophore-1 CGG CCG AAA TTT ATG AGG AA Quencher-1 WT Probe
49286 Fluorophore-2 ACC CAA ACA GAT AAT GTG ATA ACA GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47650 0.40 uM
47652 0.40 uM
49283 0.40 uM
49284 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.