Protocol 35467: Probe Assay - Sft2d2<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

>chr1:165187875-165188017 143bp GCCTCTTTGCAGTGTTTTACA AGAGCATCCTTCTCCCTTCC

Mut= 131 bp

Wt= 143 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AGCACCATTTTTTTTTAAAGATTTATTTATTATTATATGTAAGTACACTGTAGCTGTCTTCAGGTTACCAGAAGAGCCACCATGTGGTTGGTGGGAATTGAAATCAGGACCTTCGGAAGAGCCGTCAGTGCTCTTAACTGCTGAGCCATCTCTCCAGCCCCCTCGAAGCAACATTTTTATTGGCCCTTCAGaccatctctaacacaattgtgttttgtttgtttgttttcagtaagtgtgcatgacgtatgtaattgccgtttttgaaactgcaggttgtggaggcgtcgtcgctgagctggggcaccagaataaaaggcttcatcgcatgctttgctctgggaatcctgtgctcagtgctggtaaggtttcctcccacccgctgcctttgcccagctctcagctgtagccttccctttggcccagtgacttttctctgtgacactcattacatggagtggataagaaggggagggaggcagggagggcagctatgtggctctggtgggagggggtcactgtgctcaagtgcatgtgtcctctgcttccccttaggggactcttctgctgtgggtgcccaggaagggactcggcctctttgcagtgttttacaccctgggtaacatcatgtcaattgggaggtaacctttttcttttctctttgcttcaactgtgctgtttattaaacaataaaatatctaccgcgtttgagGGAGGAAGGGAGAAGGATGCTCTGTTGTTTTTAAGGATTCAGAAAGAAGGAAGGTAGATACTTGGCGTATATCACATTCATATCTTATTTCAAAAACATCTTATGTCGTGTGTTTTCTCTGTGACACCCCTGGAAGAAAGCAGAAATGGTGTCACCAGTGCTTTCTATGAGGGAA

This mutation is a 521 bp deletion beginning at Chromosome 1 position 165,187,898 bp and ending after 165,188,418 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46139 GCC TCT TTG CAG TGT TTT ACA Wild type Forward A
46140 AGA GCA TCC TTC TCC CTT CC Common A
46141 GTG GTT GGT GGG AAT TGA A Mutant Forward A
46142 Fluorophore-1 ACA TCA TGT CAA TTG GGA GGT AAC Quencher-1 WT Probe
46143 Fluorophore-2 TTA ACT GCT GAG CCA TCT CTC C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46139 0.40 uM
46140 0.40 uM
46141 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.