Protocol 35448: Probe Assay - Vstm4<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

>chr14:32863698+32863857 160bp TGGTGAAGATGACGAAGCTC TTGCAGACATACTGCCCTTG

Mut= 160 bp

Wt= 160 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 ACTTTTGAGAATGTGTCTGTTCAGATGGCCAGTGTTTGGCATGCCATGTGAGTGAGACCATCGCATATAGGAATGTGGAAGCCATAAGGCCCATAGAAGGGATTCTTAGTGGCTTCTTTGGACTCAGGATCTATGGTCTGAACTGAAGCATACAAACAAGCTAAAGTAAAATAAGAcgaaggaaggagactgaaactagaagggatatggggctctcccagtaccccaaaagatgcttcagaattccaggtgtttcctggtcatggtgaggctgggaagtatagagagtgccaagaccactgcctctgccccttcagagcatccagacgccaatagcatgtggcttgaccctggcttctgggaatggcctggctgcacaaacccaggctgcggccttcattcacctttccatcctcttccattgcagaagtctgtggtgctctcaatgtcacggtgtccccaggacctgtggtggactacttggaaggggaaaatgccaccctcctctgccacgtctcccagaagagacggaaggacagcttgctggccgtgcgctggttcttcgcccctgacggctcccaggaggccttgatggtgaagatgacgaagctccggataattcagtactatgggaacttcagccggactgccaaccagcagaggctacgcctgctcgaggagcgtcgaggggtgctgtacaggctgtctgtcctgacgctccggcccacagatcaagggcagtatgtctgcaaagtgcaagaaatcagcaagcaccgcaacaagtggacagcctggtccaatggctcctcggcgacggaaatgagaggtgagaacgctgttggcatctcggaggaggcgcagtgtgtggtagaaggtggtcctggcgttagtcagtatgtaaatgctggtcgcctcattggctctgctagtatagttagtccacgtcttgtggcctctgtgggccaaatgcctgtgagtcaagttatgaggatgaggacagctcttggacactggctgatgtggaaggtagatatggaggcagggacaacagagtgagtgactacctccgtattagtCATGTCCTGCCTGAAATATTGAACTTTGTATGCTGTCAGATAAAGAAATCTAGCCCCTACTTACTTTGGACAGAGGCGTGTGAGGGTATGGCCTGAGCTGTGCTTACTGTGGACTTGGACTGGGACTTTGTGGTTC

This mutation is a 900 bp deletion beginning at Chromosome 14 position 32,863,282 bp and ending after 32,864,181 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46085 TGG TGA AGA TGA CGA AGC TC Wild type Forward A
46086 TTG CAG ACA TAC TGC CCT TG Wild type Reverse A
46087 ATG CCA TGT GAG TGA GAC CA Mutant Forward A
46088 GTT CAA TAT TTC AGG CAG GAC A Mutant Reverse A
46089 Fluorophore-1 CAG TAC TAT GGG AAC TTC AGC CG Quencher-1 WT Probe
46090 Fluorophore-2 TGG AAG CCA TAA GGC CCA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46085 0.40 uM
46086 0.40 uM
46087 0.40 uM
46088 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.