Protocol 31537: Probe Assay - Utp4<em1(IMPC)J>-Alternate 2
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=  75 bp

Wild Type = 114 bp

>chr8:109422442+109422555 114bp GGTTCGGGCAATGAACAG CCCTTGTGCTCAGAGAAAATC

 

Sequence

Wt Sequence (deleted regions are in lower case)

TTTTTCCCAGGTCATGAGTCTCGGGGTACCGAAGCCCTGTGTTGGGCAGGAGGACAGCGGCTCTTTAGTGCTGGACTGAACGGAGAAATCCTTGAGTATGACCTACAGGCACTAAACATCAAGTACACTTTGGATGCCTTTGGAGGACCCATCTGGAGTATGACCGCCAGCCCCAGTGGCTCTCAACTTTTGGTCAGTAAGCTAGTATTGATAATACAAAATACAGTAGTCTTGATGGTCAGTCCCttccccgtctcacaagctgttttggtttgttttgaTTTTAGGCAAGTGATAGCTTTAGAGAAGAAACGCTTGTgcatgggggtattagttctgtataaaggctcatttagtctgttttctactcgtctctaatcaggttggttgtgaagatggctccgtgaaactatttgaagtcaccccagaaaaaatccagtttgcaagaaattttgatcggcaaaaaagtaaggcttgttttgggttcgggcaatgaacagcttcaacaagaggttgtctgtttgtgtcaccaaagggacaggatattttcctgtgGCTATAGGATATGCTGTGTAGATTTTCTCTGAGCACAAGGGGAGTTCTAGAAGGCATCCCATTAGGTATCCCATTAGGTAGCATAGTTGTGAAATTCAAAGTAGAAAGCTAGTGTTCCTGTGAAAATGAGTGTATTGTTTATCACCTGCATTTTAGTCCAGGTTTATGTTGTGATAAACT

 

236 bp deletion beginning at Chromosome 8 positive strand position 106,898,379 bp, and ending at 106,898,614 bp (GRCm38/mm10)

In addition there is an additional 35 bp deletion (ttccccgtctcacaagctgttttggtttgttttga) 38 bp before the 236 bp deletion

)

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
35294 CCC TTG TGC TCA GAG AAA ATC Common A
35295 GGT TCG GGC AAT GAA CAG Wild type Forward A
35296 Fluorophore-1 ACG CTT GTG CTA TAG GAT ATG CT Quencher-1 MUT Probe
35297 Fluorophore-2 CAA GAG GTT GTC TGT TTG TGT CA Quencher-2 WT Probe
37099 GGC AAG TGA TAG CTT TAG AGA AGA Mutant Forward A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
35294 0.40 uM
35295 0.40 uM
37099 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.