Protocol 45616: Probe Assay - Actbl2<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=  119 bp

Wild Type = 136 bp

  
>chr13:111255114+111255249 136bp AAGATCCCAGCCAGCAACC TGGACGACCAACCATGGAG

Sequence

Wt Sequence (deletions in lower case):

AAGATCCCAGCCAGCAACCATGGTTGACGATGAGCTGACTGCTTTGGtagtagataatgggtcagggatgtgcaaggcaggttttgggggtgacgatgccccccgtgctgtgttcccctccatggttggtcgtccacgacaccagggggtcatggtaggcatgggccagaaggactgttatgtgggagatgaggcccagagcaagcgaggcatcttgaccttgaagtatcccattgaacacggagtcgtcaccaactgggatgatatggagaagatctggtaccacactttctacaatgagctccgggtagcaccggatgaacatcctattcttctcaccgaggcaccccttaaccccaaaatcaaccgggaaaagatgactcagataatgttcgaggctttcaacacacctgccatgtatgtagcaatccaagccgtgctatccctctatgcttccggaaggactacaggtatcgtgatggattctggggatggggtcactcacactgtgcctatctatgaaggttacgctctgcctcatgccattctacgactagatctggcaggtagggacctgactgattacctcatgaagattctgacagaacgaggctataacttcaccaccacagcagagagggagatcgtgcgggatgtcaaagaaaagttgtgctacgtggccctggactttgagcaggagatggtcactgcagccgcttcatcctcactagagagaagctatgagctccctgatgggcaggtgatcaccattggaaatgaacgcttccggtgcccagaagccattttccagccttcctttctaggcattgaatccagaggcatccatgagacaaccttcaactcgatcatgaagtgtgatgtagatattcgtaaggatttatttgccaacactgtgctgtctggaggcagtaccatgtacccaggcattgccgacaggatgcagaaggaaattgtaaccttggcaccaagtaccatgaaaattaagatcatagctcccccagaacgcaagtattcggtttggattgggggctccatcctcgccagcctgtccacattccagcagatgtggatcagtaagcaggaatatgacgaagctggacctcccattgttcatagaaaatgctTCTAGGTAAGCTACCGTGTCCCTTTTCCTGGGTCATAATGATGGTACCCCACCTCACCCAGGACACTGAAGG

This mutation is a 1098 bp deletion beginning at Chromosome 13 position 111,255,161 bp and ending after 111,256,258 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
66035 AAG ATC CCA GCC AGC AAC C Common A
66036 TGG ACG ACC AAC CAT GGA G Wild type Reverse A
66037 CCT TCA GTG TCC TGG GTG A Mutant Reverse A
66038 Fluorophore-1 AGT AGA TAA TGG GTC AGG GAT GTG Quencher-1 WT Probe
66039 Fluorophore-2 CTG ACT GCT TTG GTC TAG GTA AGC TA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
66035 0.40 uM
66036 0.40 uM
66037 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.