Protocol 35468: Probe Assay - Serinc2<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

>chr4:130264184-130264272 89bp CATCCAGAACGGGTGAGG GATCTCTGGGGACAGGCAGA

Mut= 86 bp

Wt= 89 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TCTTAGTGGTTCTACTGAAGACAGGAGGGGAAACCGTTCTGGGAAGCAGGGCTCCACATGTAGCAGTATTGAAATAGGGGGTGGGTGAAGAGGGGTGGGGGTGGGGCGGAGAGCATCAGGGAAATGATCAGCTAGGCGGGAGCCGTTTATGGATTAGGGGCCTGGGTGGCCTTTTTTAGTGTTTAgagcaacacggggtgggaaacaggccttagagatggaggcaggctcagacctgatggcagactgcagggttctggtgggtgggacccctcaatcctccctgttgtctccacagctgccctgggtgtgtgaggacaggacccagcaacccctggtcctgcaggggcccctggactgcggctccctgctgggtttccgcgctgtctaccgtatgtgctttgccacagcagcctttttcttcttcttcatgctgttaatgatctgtgtccgcagtagccgggatccgcgagcagccatccagaacgggtgaggggcggccacctacaccctggcacactcccctgGGGGGGGGCTTTTGTCCCCTCTGCCTGTCCCCAGAGATCTCTAGACTCCATCCTCAGACTCATTAGAAGCCAGAGGTCAGCCTCGTCTATCCCTAGGAGCCGCCCACACCTTTCATTTTGTGAAACAGGGTATCTCATCTGGGGCTGATTCCACTAGCCTGGCTAG

This mutation is a 342 bp deletion beginning at Chromosome 4 position 130,264,223 bp and ending after 130,264,564 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46144 CAT CCA GAA CGG GTG AGG Wild type Forward A
46145 GAT CTC TGG GGA CAG GCA GA Common A
46146 GGA GCC GTT TAT GGA TTA GG Mutant Forward A
46147 Fluorophore-1 ACC TAC ACC CTG GCA CAC TC Quencher-1 WT Probe
46148 Fluorophore-2 CTT TTT TAG TGT TTA GGG GGG G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46144 0.40 uM
46145 0.40 uM
46146 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.