Protocol 34652: Probe Assay - Sfswap<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

>chr5:129503672+129503759 88bp ATTGAATGGTTGACCAAAGC AGAGCTAAAGGCAGACAATCC

Mut= 84 bp

Wt= 88 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TGAGAGTTCGGGACTAGCAGGAAGAAGCGAGGAAATTTCGCCCGTTTGGTTCTGTCGGATAGGTATTTATGGACTCGGTCTGTGTGAATGTAAGCTGTAATATTTAACTACTGAGAACAGATATTTTTTTTGCAAGTGGCATTGAATGGTTGACCAAAGCATACATGGTAAGAACTGCTAGcaagggggtacgttttctgctctaaaggattgtctgcctttagctctaagttctgttgtgctgtagagaggtgcttagattttgttatgattactccagtttaaaagtaggagagagatgactaggcatattaagtataaatctgaagctttgcatttgttttaatatttttatttcattagtttaattggtaagtatgtgcatagagtgtcacacacaaccagcatgggtgtagacttccctccagtgaccagcctgttcacattacagatacgacgggcgtggtcacctgcatgacctctctgcgtacgatgctgagtacgccacgtggaaccgagactaccagctgtctgaagaggaggctcgggtagaggcgctgtgtgatgaagagaggtatttagccttgcatacggacttgcttgaggaggaggcaaggcaaggtactgctcaagaaagacttacttcagccacacctttttaaaattttaagtatttaaaaagttactcccgttcatttttatactcactctttctgatattatcttgacagtaccCAGTGGATTGGAAAAACAGGAGTCTTTGCGTTCTGAGAGGACCTCAGGATAGTTTATATGTAGAGCCACAGAGAATTTTCCCAGCTTTTGAGGGCAGACTGGGATTTGAAAAAACACAAAAACAAAAAACCAAAAAACAAAACTCTTTAACTGTTCTTCTTTAACAGTGTT

This mutation is a 554 bp deletion beginning at Chromosome 5 position 129,503,713 bp and ending after 129,504,266 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44152 ATT GAA TGG TTG ACC AAA GC Common A
44153 AGA GCT AAA GGC AGA CAA TCC Wild type Reverse A
44154 GGT CCT CTC AGA ACG CAA AG Mutant Reverse A
44155 Fluorophore-1 AGG GGG TAC GTT TTC TGC TC Quencher-1 WT Probe
44156 Fluorophore-2 CTG CTA GCA GTG GAT TGG AAA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44152 0.40 uM
44153 0.40 uM
44154 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.