Protocol 34781: Probe Assay - Spats2<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

Mut= 101 bp

Wt= 99 bp

>chr15:99173271+99173369 99bp TATTCCTGGCATGACGGTTG AGCAGACTACAGCAGGGTGA

Sequence

Wt Sequence (deletion in lower case):

TCTCTTGGTCCCTTTTATTTCTATAAATGCTCTGTGTTTTAAATTTCAAAATTAAGTTTTCATGTGGTGCTACTATTTTTCTACTTACTATTCCTGGCATGACGGTTGGATGCGTTCTTCCTGACATTAAGCAGTGATCATATTGTGAACTgcactggcactattattcaccctgctgtagtctgctgcccggtgacctgcctgtgccctcttttgcagataaacgcggtacgtgcgatagttcccaataagagcaacaatgagatcatcctggtcttgcagcactttgacaactgtgtggacaaaacagtgcaagcgttcatggaaggtaacgccagttccctcagtgtagactctggaacctgagccgcagcgcagtctccggacggcgctgcatactgtggtcttctgttagctagggtactcctttcattcccaagctatagaaaactggaagagatgctaagaaaactcctttgctgtcagatatgttctccccctccccaaatcaagaaactgtgcagctgacagactgtgggatttgaatGGAATTGGATAAAGTAATTGAAGACGCTATTGCAAAGGAGGAAGTCAAGTGGCCCACGAACACAGACCAGTTCAAGACCCAGCATTGATTTCCAAGCTCCATTATGAGTTAGTTTGGTTCTGGCAGGGGACTCAAATGTTATGAGTTCAGTTTCCTTACATTTCTATATGCTAGATAGAAAAAAGTAACAGCCTTGTCCAACTCCTTAGATTTATAGAGTTAATAAGTGCTTCTTAAAATTCTTTCCATATAACTTGTTTGAAAAAACTTTACTCTGAAGCTGTTTTGCCTTTGAACAGAGTGGCCAGGGCTCCTTGATGCGTTTTCCTGAATCCGCAAGCATAGAAT

 

This mutation resulted in a 396 bp deletion beginning at Chromosome 15 position 99,173,334 bp and ending after 99,173,729 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44431 TAT TCC TGG CAT GAC GGT TG Common A
44432 AGC AGA CTA CAG CAG GGT GA Wild type Reverse A
44433 CCT TTG CAA TAG CGT CTT CA Mutant Reverse A
44434 Fluorophore-1 TGA ACT GCA CTG GCA CTA TTA TTC Quencher-1 WT Probe
44435 Fluorophore-2 TGA ACT GGA ATT GGA TAA AGT AAT TG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44431 0.40 uM
44432 0.40 uM
44433 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.