Protocol 47914: Probe Assay - Ttc27<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

Mutant=  76bp

Wild Type = 89 bp

>chr17:74747859+74747947 89bp ATTGTATGGCTTGTGAAGGT GGAGAAGGAGCTACAAACTC

Sequence

Wt Sequence (deletion in lower case):

TTCAGGAGGCTGAGGCTGGTGGACCTTTGTGAGTTTAAGACTAGCTTGGTTTGCCTAGTGAGTTCAGGCCAGCCAGGGCCACATAGTGAGACCCTGTTTTAAATATCAACAAGAGACAGAACACCAAGCCAAACTAAACAAAAACTCCAAAGAAAATCAAAATAAAAATAATGTACCCCAACCTCCCTCCAAAAACCCCAGCaagcatcaatatcatttccattagcagtctattgaattaatgaatgaataaatgaatgaaagaatgaaagaatgaaatgtaactcttgttctaaatattttcttttgtgtcccactagaatcttgagctcaatgatgatactgttctgaatgaaataaagttagcagatagtgaacggtttcagatgccagacctgtgtgcggaggagcttgccgtggtccttggagtctggtacgttaatgtctgacttggctgtgttgaacgctttgactgttgtcccctgagtttgggaagatcttcatcaaaacctccttgtacagtaggtattgtatggcttgtgaaggtggagacttcttgggcaacgatttaaaggaTCAGAGGTTCAGAATCTATTGAGTTTGTAGCTCCTTCTCCTAGTCTTTGGTTATCTTCAACTAGGTGTAGATTTTTAATCAAGTCCTAATAGACGAGAGTGATTGTGTGTGGGGGATGCATGGAGAGGCAGAGGTGGTCTGTGGATGTTCACACCAGTGCTCCCCACCTTATCTTTCGTTAGACAGGATTTCTCACTGAACCTGATGCTCACCTCTGTGTTGCCTATAAAAGGGATCCCTTCTTTGTCCTCATAAGACTAGGTTACAGAT

 

This mutation is a 374 bp deletion of region Chr17:74,747,534-74,747,907 (GRCm38/mm10)

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
71604 ATT GTA TGG CTT GTG AAG GT Wild type Forward A
71605 GGA GAA GGA GCT ACA AAC TC Common A
71606 AAT AAT GTA CCC CAA CCT CC Mutant Forward A
71607 Fluorophore-1 TTG GGC AAC GAT TTA AAG GA Quencher-1 WT Probe
71608 Fluorophore-2 AAC CCC AGC TCA GAG GTT CA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
71604 0.40 uM
71605 0.40 uM
71606 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.