Protocol 37255: Probe Assay - U2af2<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

>chr7:5070062+5070154 93bp AAGCCTGGGCTGTCATTG AACTCTGACAATGTTGGAAAGG

Mut = 93 bp

Wt = 93 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

ATTGCCCATAAGAGTCCACTTTGATTTGTACTCTTGGAGACTAATGTCACTTCATCAGAAAGCCTGGGCTGTCATTGCCACgtaacttagttgacctaacttttctatgctggcttctcagcagttggtccctttccaacattgtcagagtttatgatagtgtgcttgctatctgctgggtgcgaggatattgctttctagagtgtgagcttctagaagctatctggtagtgcttgttcccattgttcattatgggtttggtactatgtttgtttatataagtgggaggaaagttaagagtcttgggggctggtctagtcccaacctaccccttagctctgttgtttctcttccttcagttccggtcagtggatgagacgacccaggccatggcatttgatggcatcatcttccagggccagtcattgaagattcgaaggcctcatgactatcagccattgcctggcatgtcagagaacccctctgtttatgtgcctggtgagtgggaattcgttgggtgggtggtatggggagtcactgtttcctcaacccaggttcctgtacttaGTCCAGCTAGAGTTGCTTTGGAGATAGTGTGTGTGCGCTGTGGATGTTCTTCTAGCTTGGCAGGTGTCCTGGGCCTTTGAGGTGGTGATCTGTGATCTTCAATCTTAAAGTTTAGCTTAGCTTGTAAATGTCAGAGCCAGGTCCAGATCAGAGGTGTCCTGGAAAGTGTGGGACTTGCAGTCTGGGCATTGTTTTTACCTTGTCCCCTTAGCCCTGGGATTTTAGGGCTTGATGTGTTGT

This mutation is a 486 bp deletion beginning at Chromosome 7 position 5,070,084 bp and ending after 5,070,569 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50900 AAG CCT GGG CTG TCA TTG Common A
50901 AAC TCT GAC AAT GTT GGA AAG G Wild type Reverse A
50902 CAG GAC ACC TGC CAA GCT A Mutant Reverse A
50903 Fluorophore-1 CTA TGC TGG CTT CTC AGC AGT T Quencher-1 WT Probe
50904 Fluorophore-2 ACG TCC AGC TAG AGT TGC TTT G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50900 0.40 uM
50901 0.40 uM
50902 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.