Protocol 32224: Probe Assay - Als2cl<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

  

>chr9:110884741+110884839 99bp TTCAGCCCTTTACCCTAGCA GGCGTTGATGGGTAGTCCT

Mut= 98 bp

Wt= 99 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence: (deleted region in lower case)

 

TTTGACCTTGGGCAGGTGTTTTCCTCTCTCACAACCTGTCTGTGTATCTCTAAGGTGGTTGGGAGTGACCCTGAAGACTCCACGTTCTGCATCTGGGTCTAAAGAGAAGAGGCAGAGACTCAGGGGAGCTAGAAATCTTGGTTCCCCAAAATGATTAGGGTTGTCCTGTTAAGCAGGAGGCACACATACAAGCCTCATTACTCAGGGTTCAGCCCTTTACCCTAGCATGCCCTGCCCctgaggagctctatggggtactaatacagtcctgagcctcctagagctacaggactacccatcaacgccagctcagggtggctctgctttccacagacccggagccctcagatccctgtggcaaagagtgccttcggcttctgcagcagctgcatgagagtgcccagcggttgtggtatgtgacagagcaaagcctgctttcgctgcggcagaggctataccacccaccttccaagggtctagaggccgtgctgttactgagcaatgctgaccatgtcctacaagcccacatggagtaagacgccctgctctcacccctgcctttggacagtttgttgcttggccacagtgccagagtgccatctctgctatttgatcctaagggtcagctggggtgggtctcctatggtctagggtattggcttggtagcaggcatataggctcaggcctggaccagacctatgagatacaactgttgggtcatggcttgagggcaactgcctagaccatcaggattCTGGCTGCTCAGTGGGTGGTATCTGGGACCCCCCAAGAGGGCAGAGTCTGAGTGTGCATTGGGAGCTGGGCTTTCAAGGTGATCCTGGGTTGCTCATAGATACATCAAGTCCTACACTGATTGCGTGGTGGCGCAAGCTTTTCAGAAGGTCTCAAAGAAGAGGAGG

This mutation is a 518 bp deletion beginning at Chromosome 9 positive strand position 110,884,771 bp and ending after 110,885,288 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38635 TTC AGC CCT TTA CCC TAG CA Common A
38636 GGC GTT GAT GGG TAG TCC T Wild type Reverse A
38637 CAG CTC CCA ATG CAC ACT C Mutant Reverse A
38638 Fluorophore-1 AGT CCT GAG CCT CCT AGA GCT AC Quencher-1 WT Probe
38639 Fluorophore-2 CTG CCC CTG GCT GCT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38635 0.40 uM
38636 0.40 uM
38637 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.