Protocol 33159: Probe Assay - Safb2<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

>chr17:56582929-56583016 88bp TGAGCAAGAGGGCTGTAAAGA AGCAGAGCAGACACCCATTC

Mut= 90 bp

Wt= 88 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TAAAGGTTTGCTTTGTTATGGGAGGCCTTTTGAGCGCCCCATTCAGCTCTTTGCCCTGCCTGAATCAAGGACAGGTTTCCTACCATCATCCCTGTGTTTGATGAAAGAGCACACAAGATGAGCGGTCACCAACCCTGATTTGGGGTTAATACTTGGATTTTATTCTATTTTTTTGTCTTGGGTTGGGGATTACATCTAGGGCTACAAGCTGCCAGGTCATGTATGACCACAACCTGctgacagtgactctgcaacccaagctttcagcttccctttctctaggttttgggtcttgagcagggaaggggagctggctgctaaatgtggtcttaagctagattgtgaacacttgcaactgcaaagttgcttcttaaagaacactttgtgtcaacccctcctaaaagataactttatttaaaacttcatactctttcccatcaggcatttaaggaggaaggacaagaacctgaggaggttggaatcagctggggagctgtgagcaagagggctgtaaagagaaacaccaaaggtacttaactgcctgtgcttgacgCTGGGCCTGGGGAATGGGTGTCTGCTCTGCTCTGTGTATGTGTAATATGCATACTTAGATGTGCATGTGTGTGGGACAGCCATAGTGTCACATTGTGTCCACCTTAGGTTTTGTTTTTGTCTTAAGGTTTATTTTGTGTGTGTGTGTGTGTGTGTGTGTGTTTGTGTGCGCGCTTGCCTGCATGTGAATGCACATGTGTTTGGTGCCCACAGAGGTCAGAAGCAGGTCTTGGACCCCTT

This mutation is a 317 bp deletion beginning at Chromosome 17 position 56,582,960 bp and ending after 56,583,276 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40784 TGA GCA AGA GGG CTG TAA AGA Wild type Forward A
40785 AGC AGA GCA GAC ACC CAT TC Common A
40786 TTG GGT TGG GGA TTA CAT CT Mutant Forward A
40787 Fluorophore-1 CAA AGG TAC TTA ACT GCC TGT GC Quencher-1 WT Probe
40788 Fluorophore-2 ACC ACA ACC TGC TGG GC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40784 0.40 uM
40785 0.40 uM
40786 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.