Protocol 34031: Probe Assay - Zfp438<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

>chr18:5213099-5213198 100bp AGTGAACTGCAGCCAGGAG TCCCTTCCACGAGCACTTAC

Mut= 121 bp

Wt= 100 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AGTGCAGGGAAATGAACCCAGGTCCTATGCAATGCAGTGTGTCCTCTTAAGTGTTGGGCCAAGTCTCTAGCCCAGGTCAGATGTGACTTGAAGTTTCAGTCCAGAACAGATACATTGTAGTAATCTATGTATTGTCATTGCAGGTGAATCAAACATCCGttctggaacaacacagagtgggaaaacattacagagtaagagtcagtttaggaccattgcaccaaaagttgtacctaaggtcctaacatccagagtgcttccctgcccttcctcaatttctgaccaagggagtctgactctaatgcccaagcctctgggcatgcccacccagaactatgctctgatgcaggtggctggccaagaggggacgttttctcttgttgctttcccaaatgttgcctcagcccagccagttcagaagcccagaatgtccacacatgaaaaccttaaactgcctattcctcgataccaaccactaagtagccaaggattaagaaagaaacaggacctgagctctcccaagagtggctccagtgaccctcctggcccatcccagctatgtcatttgacctcttccccacaagcctatcctgatcctcatcacaaacctagcctgtggaagcaaatgccaactttaaacccttctcccaccaacatcaacacagctacactgaccagtggtgttggccaaggagatttgagtcctctagtgaccaacagctgtggcgatcttgagctttctgccatgccagtgtacactacagaggactcttcttcccaacagagcctcccagcaagtatgcagaaggcaggttgtgccaggaaggaggcacccactacacctgctattgctagtgaagaactcaaagaacagttagcttctgcacagactgtcttgagcagtgcagttcagttgctctctgtagtacccaagggtaaactgcccatcctgcccttaccaagagtgaaaactacagaggtgggcaaagtggaatcagatgctgataatgcagaattgtctttatctggatgcagagcaaactgtgatgagagactgtccatcacagaagggtttgatgcagccacagaaatagccagcaagatagctgctttgcatgggtctaagcagagtgcatgtgaaagtgccttctgtcctgtcaccaaattggatctcagccacaaggcaaaacccagcagtggaataaagagaagaggaagaaaatggaaagtgccagatgagattttagcattgcagggcaaaaggaggaaatgcattattggtatgtgtggagatggcatagaaagagcaagaaacagtccccaagaagccagagaccagaagcccagggttgcttcaaggaagtatcgtaacatcatgcccaagcctgtccttgtcctgtctgctttggcacccctggcgtctcatacggctgtgctgtctcaggctcccagcagcctaggtcaagatgttctgaataatgccctgccttcaaaatgtcttggctccaagcaaagtgacagctctacccctaagcccagctctgtactccgaaatggattctctggcattaaaaagccctggcacatgtgtccagtctgcaactaccactttcaattcaaacaccaccttctagaccacatgaatacacacaccaacagacggccttatagttgcgggatctgtcgcaagacctatgtgcgccccggcagcctgagtgcacatatgaaacttcaccatggtgacaaccaccccaagaaactagtgtgctgtgaattttgtgcaaaagtatttggtcatgtccgagtctatttcggccatctgaaggaggtgcacagggttgtgatcagcacagagccctcccccagtgaactgcagccaggagacacaccaaaaaacaagaacagagatcctggtATGCAAGGACCTGACAACTCACTGGAGAGGTAAGTGCTCGTGGAAGGGAAGCTGACAGGAAAGTCTGCTGTAATATGAGAAGGAAGACCAACTCGTGGATATTTCATTCCTCCCTAGCATAGGGAATAAAATATCCATGGAAGGAGTT

This mutation is a 1756 bp deletion beginning at Chromosome 18 position 5,213,148 bp and ending after 5,214,903 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42679 AGT GAA CTG CAG CCA GGA G Wild type Forward A
42680 TCC CTT CCA CGA GCA CTT AC Common A
42681 TTG AAG TTT CAG TCC AGA ACA G Mutant Forward A
42682 Fluorophore-1 ACC AAA AAA CAA GAA CAG AGA TCC T Quencher-1 WT Probe
42683 Fluorophore-2 CAA ACA TCC GAT GCA AGG A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42679 0.40 uM
42680 0.40 uM
42681 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.