Protocol 31800: Probe Assay - Stk33<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=  136 bp

Wild Type = 144 bp

>chr7:109331313-109331456 144bp CTGTGTTCCTCACCATCTCTG CTGCATTGTTGCTAACTAAAACGTC

Sequence

Sequence (deleted region in lower case)

 

GGCGCGGTAGAAACTTGTTCTAACCTTCACTACATTCCTCTTTCCACCCCGCAGCTTCCAGTACTGTTGGTACAGATCTACATGTTATTTTAATGCTGCTCCATTCAGGCTTTAGGGGTTTTGTGTGCTGGCCACCAGCTCTGCCTGGTTTTCCTCCACTTTCCACCCCCAGAAATGACTGCACACACGTTCAGTCTCAACTCTTCTCGCTCAGTGAGCATTCTTTATTTAGAGGCTGCCTCTCTTCCACAGAcccatggcatgctggctgttagcatatatcacatgatattgtaactctgctgagcttcctgaggtcatcgctatgtcgcttggccctctgctcctcgtgtattcaggacagtagcttatatagagtagataagacattttcacattaataaattagaattgtgagcatacttttgatatctaacacatggaacttcataactttctcttgttatgcctttaaacctgtaatgtcagaaaatgtatctcgtgatggagctttgtgaggatggagaactcaaagcagttatggatcaaagagggcacttctcagagaacgagacaaggctgataattcaaagtcttgcatcagccatcgcatatcttcataacaagggtaagacaagaactgtgttcctcaccatctctgtaacaggaatacatcactgttgtacattggtagagcttggtaatgcAAGAGGAGAACAGGTACATTTTCCATAGTTTCCCCTTTTAATTGTATTTATAGACGTTTTAGTTAGCAACAATGCAGTCATAAAATCAGACTGTCCGTGAAAACGTCATGATCATTCTTATTTTAGAAGTTTAAAATGGGCTAATATGATCAAAATTAAAGTAGAAAAATCAAAGTGAATTTTCCTAAAATATTT

454 bp deletion beginning at Chromosome 7 negative strand position 109331843 bp and ending after 109331390 bp (GRCm38/mm10)

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37688 TCT CAA CTC TTC TCG CTC AGT G Mutant Forward A
37689 CTG CAT TGT TGC TAA CTA AAA CGT C Common A
37690 CTG TGT TCC TCA CCA TCT CTG Wild type Forward A
37691 Fluorophore-1 TTT AGA GGC TGC CTC TCT TCC Quencher-1 MUT Probe
37692 Fluorophore-2 TGT TGT ACA TTG GTA GAG CTT GGT Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37688 0.40 uM
37689 0.40 uM
37690 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.