Protocol 35452: Probe Assay - Ubac1<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

>chr2:26008521-26008660 140bp CCAAACTCAGGAGCTCTTCATC CACACACCCCAAAAGATCATA

Mut= 145 bp

Wt= 140 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GGTTCTTTGTTAGTTTTTGAGACAATAGCCTCACTGTGTAGACGAGGCTGACCTTGAATTCACAGAGATCTGCCTGCCTCTGCCTTCCAGCTCTGAGATTAAAGGTAAGGCCCAATATGCCCTTGTAGATTTGGAGGGCTATGTCTGGGATAGTGTTGCAGATTTAAgcaagtttgttggacattctgagaaataatctttttggtttgtttgatttttgagacaggtttttactgtagaattaaggttggcctcaagctcccagcagacttcttttctcagcctcgcaagagcagggattacaggtgtgcatcactgtaccgggcatgatctctttctttatcctactgcagttccagacagaactccggaagatcctcgtgtctctcattgaggtggcacagaagctgttagcactgaatcccgatgcagttgaattgtttaagaaggcaaatggtatgcgtgggtcctaggctgctcctaaaagtgtccccagccctcacactgaaagcaagttctgctttggttaatgggggagagggtttggtgtcagtaattgctaggtgaggtgagcctctactgatgaggctggggggcttcaaaggactctggaatcccctcatggcaggccttgtctttctcctcagctatgctggatgaagatgaggatgagcgagtggatgagactgccttgcggcagctcacagagatgggctttcctgagagcagagcctcaaaggcccttcggctgaaccagtatgcttgctttcttcttgccctgggcttccctgtgattctccttccaatctctggagtcatacttttttttttaatttttatttatctgtgtatatgtacatgtgtgtatgggtgcccatagaagccagagaggaagttggatcttctggagctagaattacagactgctgtgaactgcctgacacaggtgctaggaaccaaactcaggagctcttcatcttaatctaggagccagctccccgatcccattgagtcatttctaattgggtgttctagtagagGCACCTTATTGCTGTGCTTCAGAAGTGCTTTCATATATGATCTTTTGGGGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGGAGGGTCTCTGTTACCTATGCTGGACTTGAACTCCTGGACTTATGTAGTCCTCCTGCCTTAGGCTCCTGAGTAGCTAGGATTGAAGGTGTGAACCATGATACC

This mutation is a 873 bp deletion beginning at Chromosome 2 position 26,008,577 bp and ending after 26,009,449 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46095 CCA AAC TCA GGA GCT CTT CAT C Wild type Forward A
46096 CAC ACA CCC CAA AAG ATC ATA Common A
46097 TCT GCC TTC CAG CTC TGA Mutant Forward A
46098 Fluorophore-1 CCG ATC CCA TTG AGT CAT TTC Quencher-1 WT Probe
46099 Fluorophore-2 TGT TGC AGA TTT AAG CAC CTT ATT G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46095 0.40 uM
46096 0.40 uM
46097 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.