Protocol 31665: Probe Assay - Srbd1<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= 85 bp

Wt= 100 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence: (deleted region in lower case)

 

CATTTTTTATTGTTTTGACATTCTCTAGGTAATAAATGTTATCAGTTTAGTAGACTTACTTTTTTGTTATATTATGTGATTCCTCTTGTCATTTGTCTTATATTTTTTAACTGCTGAGCCTTTGACTATGGTAAATATTTTTAGTGGCATAGTGGCTAATCATTGATATTGCGAGAGGATGTCTGCTTTGCTTTAGGAGTATAGATTATTCCTAAGTCACTAAGTTGATTGGCATTTGATTGACTCCTAACTTTGTGAAGACACTTTAATGTTATGAAAAGATGTGGAAGAGAGAAAATTTCTTATATTTGAGAAATAGTACAACAGATATCTTTATAATCTGTTGACTGGCAGGATCACTATAAAATAATACTTGCAGAATACTCCATCCTTTAATTCTAAAGCAGACTTATTGCCCTGGAAGGGCTTGTCTTTTTCAAGAGATGGTCTCAGCGTCATAAGGTATTTGCACATTGGAAGGCCACTGGGTTCTCTTCTGACCAGCAGGTTATATCCTgtggggagccagtgtttgggcttgtgaatgagccatatatttttctcccctctatggcatttctgacagcagagctttggagtattagttttaaatggacgaaatgataaacctactcatgtcatgcggactctgatttgtgttccgtttttccccagatgccctcattgcgcaaaagagtgaaaatacaggccgagaatgcagaacccaaagatgaatgctctttcttctctgagttgtaagcatattagtgtaatatgttgttgggggaatggtgactattttaagagtaatGTGGTCTGTCCCGTAGACACTTAAGTCATTTGGGTCGAAGACATGTGTGTTCTTGTCCTTGCTCTATGTAACAACTCTCTGTTATAATTGTGCCATGATCCAAGAGAGAGAAAGCAGCTATCAGGTTATGTAATGTTTACCCTGTACAGGTGAAAGAGGGCAAGCTGTTGTTTACACTGCTTTGGAGCAGAGCTCTTTCTTTTTTATGATATTTTTCATGTGCATTGGTGTTTTGTCTGAATATCTGTGTAAGTGTGTTGGATCCTGAGGAACTTGAGTTACAGATGGTTGTGAGCTGTCATGTGGGTGCTGGGAATTGAACCCAGGTCCCGGAAGAGCAGCTAGTGTTCTTAACTGCTGAGCTATCTCTACAGCCCCTAGTTGTAGCTCCTAACCCATTTAGCATTGCTACTGTGTATAGGGTCACCTTTTGTTTTGGGAGCTGCCTGATGCTTTATAGGACATTTAGTGGCATCTGTGGCCTCTACTAAATGTGGCAACAGTTCTTGATATGACAACCAAAAATGTCTTTAGATAATGCCAAATGTCTCAGAGGCTCAAAATCATCCTCTGCT

This mutation is a 294 bp deletion beginning at Chromosome 17 negative strand position 86,142,597 bp and ending after 86,142,304 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37360 GCC ACT GGG TTC TCT TCT GA Common A
37361 GAA ATG CCA TAG AGG GGA GA Wild type Reverse A
37362 ACA CAT GTC TTC GAC CCA AAT G Mutant Reverse A
37363 Fluorophore-1 AGT GTT TGG GCT TGT GAA TGA Quencher-1 WT Probe
37364 Fluorophore-2 TGG TCT GTC CCG TAG ACA CTT A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37360 0.40 uM
37361 0.40 uM
37362 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.