Protocol 33276: Probe Assay - Tesk2<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

>chr4:116750971+116751094 124bp TCATATTTCTGCATCAGCATCC AAATAAAGCGAAAGCAGCAG

Mut= 112 bp

Wt= 124 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

CAGTGCTCTTGCATTTTGAGCCATCTCAGCCCCCCCCCCCTTTTTTTTTTTGAGACTAGATCTCACAGTGTAGCCCAGGCTGGCCATGTACTCATATTTCTGCATCAGCATCCAGAATGCAAGATTTATTTTATCTAAGCTGTTACTAGACATCCTGGTatgttctgtgtgtggagaccttgctgtcacctacagctgctgctttcgctttatttaactttgtaggtgcgacacagagcttctggtcaggtgatggctctcaagatgaacacgctgagcagtaaccgagcaaacctgctgaaagagatgcagctcatgaatagactctcccaccccaacatcctcaggtaatgggttttccttccttccttccttccttccttccttccttccttccttccttccttccatccaggatcagtaagaggcatcactggatgcctgctgcagtagtactttatctcatggagtccatcttGCTGGGGTTGAGCTAGGCTCTAGGTCTTTTTCTCCCCATTACAACTTAATTTCCTGTAATTAACAATATTTAGTAAGGCAACTTCTCTCAAGTTGCTTTATTCTCATCAGAACATCAGTACTAGCCCATTAGAATAATTGTTTCCTACTGT

This mutation is a 319 bp deletion beginning at Chromosome 4 position 116,751,039 bp and ending after 116,751,357 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
41023 TCA TAT TTC TGC ATC AGC ATC C Common A
41024 AAA TAA AGC GAA AGC AGC AG Wild type Reverse A
41025 TTG TAA TGG GGA GAA AAA GAC C Mutant Reverse A
41026 Fluorophore-1 TGT GGA GAC CTT GCT GTC AC Quencher-1 WT Probe
41027 Fluorophore-2 CAT CCT GGT GCT GGG GT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
41023 0.40 uM
41024 0.40 uM
41025 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.