Protocol 34790: Probe Assay - Secisbp2l<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

Mutant=  132 bp

Wild Type = 133 bp

>chr2:125773330-125773462 133bp AGCCCAGCCCAAGTCCTA GGATGATAGAAGCCCATAACTTG

Sequence

TGGGAAACAATAAATGGATGTGGAGTGTGGCAAGCTCTCAGGCTGGATTCTCTTACGATGCTGTGCTCATGGATATTACAAGTGCAGGttatcagcaaaaaaagctgatggtgtcagtggtgcctctccctaggagtattgtgaaggcactgctctctgtcttattgtttgttctgctttgatattactcttggatgtcaatagatttttttctctctcttgtttggttttgcagacaatttcctttatataacaacgatatacgatggcagcagcccagcccaagtcctaccggaccctaccttgcctaccccattatatctgctcagccacctgtttccacagagtacacctattaccagctgatgccagcaccctgtgcccaagttatgggcttctatcatccttttcctaccccttactccagcaccttccaggctgcaaacactgtcaatgccataagcacagaatgcaccgagcgtccaaatcagctggggcaggccttcccgctgtccagtcatcggagcaggaatggtaacagagggccagtggtgccaaaagtgagtggccacactgtgtgtgtagtttttaaatatgctatttctgttgttagtactagtataggtaatttactacttggaccattggccTTTAATGCTTAATTTGTCACAAAGTATTCATCACTAATAAAAAGTGTTGTAGGTATATTATTTAAAAATTTTTTTTCTTGTGATTTATTACCCCTCTTGTTTGAAAGTGTAATTTCAAGTCATCCTCATGATCATTCAGAGAGTTTTACATAAACATAATTTACAATTATTTCTAAATTATTTTGATTTAGTGCTAAAA

This mutation is a 562 bp deletion beginning at Chromosome 2 position 125,773,086 bp and ending after 125,773,647 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44462 AGC CCA GCC CAA GTC CTA Wild type Forward A
44463 GGA TGA TAG AAG CCC ATA ACT TG Wild type Reverse A
44464 Fluorophore-1 CTC AGC CAC CTG TTT CCA CAG Quencher-1 WT Probe
44465 ACA AGA GGG GTA ATA AAT CAC AAG Mutant Reverse A
44466 CGA TGC TGT GCT CAT GGA Mutant Forward A
44467 Fluorophore-2 AGT GCA GGT TTA ATG CTT AAT TTG TCA C Quencher-2

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44462 0.40 uM
44463 0.40 uM
44465 0.40 uM
44466 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.