Protocol 36974: Probe Assay - Spg11<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

>chr2:122112065-122112182 118bp TGTTGGTTGCATGTTGAGAC AACAAGCCGATGCACAGTTC

Mut = 139 bp

Wt = 118 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 TTTATTCTAAGTGTAGGTAAGATTTTGGTGACTTACAAGAAAACAAAATATCTGAGCTTTTAATGTTGGTTGCATGTTGAGACAACATTTGGGTATGGCTAACGTGTAATGCTAGATTGGGTTTAcccttctgtaacaggatggtaagcagcagctaggaagaactgtgcatcggcttgttaagccgctggcttctcagtcatctggttactgtgttggagaatgccctctttccagccctcctgctgtacctgcgaggtggtaaccattcagacaaccttatgatttaaagggtcctgcgtatttataaactgacacttctttaactctaactcagatatttttaacactatggatggcacgcaggtagcgcaagtggacctggctcttctcagagaagattctgagcagctggagcccgccagtgtctcttcctttacatcgctgagagtgtctcaggacttggatgttctggtgctggtcagctcagccagtactgcagttgctcttaacctacatttgtatttcaggtaggtagatggctcaaatttctcatttgaggtaaatagacccataaaaggtttaaaatatactccttagctttttttaaaagcctctttgctaTGCAGTTAGATCTCATTATGGCTCCTACAGTAAGACTGTTACCATGCAAGAGTGTGTATAAATGATTGTGGCTGCCTTTATATTTTGGCTTGTATATTAAA

This mutation is a 499 bp deletion beginning at Chromosome 2 position 122,111,622 bp and ending after 122,112,120 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50154 TGT TGG TTG CAT GTT GAG AC Common A
50155 AAC AAG CCG ATG CAC AGT TC Wild type Reverse A
50156 AGG CAG CCA CAA TCA TTT ATA C Mutant Reverse A
50157 Fluorophore-1 TCT GTA ACA GGA TGG TAA GCA GCA Quencher-1 WT Probe
50158 Fluorophore-2 TTT ATG CAG TTA GAT CTC ATT ATG GCT CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50154 0.40 uM
50155 0.40 uM
50156 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.