Protocol 37820: Probe Assay - Tigd3<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

>chr19:5893009-5893101 93bp CATGGAGCTGAACACCAAGA TGGGACATCTTAGACTCATCCA

Mut = 91 bp

Wt = 93 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

CTTCCCAGGGAGTCATGGAGCTGAACACCAAGAAGAAGCTTCACGCCCTgtccctggctgagaagatccaggtgctggaactcttggatgagtctaagatgtcccagtcagaggtggcccggcgcttccaggtctcacagccccagatctcacggatctgcaagaataaggagaagctgttggcagactggtgcagtggcacagccaaccacgagcggaagcggaagcgtgaatccaaatacagtgggatcgatgaggctctactgtgctggtaccacattgcccgagccaaggcctgggatgtaacggggcccatgttgctccacaaggccaaggaactggccgatatcatgggccaggatttcgtgcctagcattggctggctggtccgctggaaacgtcgaaacaatgtgggctttgggactcgccaggtccttgtccctctgttccctcctgaagctcctcctgcagttctcccatctcaggcccagccacctctttctcttaaagacttctcaccagaagatgtttttggctgtgctgaagtacccttgttgtatcgcgcggtgcctggcagagtgtttgaatgtgatcggttgcaagtactgttatgtgccaacagcagaggcacagaaaagcgaagggtatttgtgggtgggctccaggcggccccaagatgcttctttggggtgagcagtgaggcactgcctacctcttatcaccctgacctagccattccctggtcagagtggctggcacagtttgaccaggacatgggacagcagggccgacaggtagccttgctactggcttctggagtggtagaggaatgggccagccttcctggactccatcacgttcgactcctgcctctctctgcctccagcaccactccttccctgcctggctctgtgattttggcctttaaggcccattaccgtcaccgtctactgagcaaactggctgccatgcagagtgggaaagagggcacctccctggcggaggccagggctagcatcacagttttggatgctctgcacatggtggccgcagcttgggccaaggtacctcgccagctcattttgagcagctttgttcaagaagggctcgctccgggcaaaacaccactgtccctggacaaagacactgagatgtcaccagtccctagtgggctgagccaagaggagttttctcactttgtggacctggaggatgaggacccagggcctagagtgtgcaaagaggagacgggtactgaagacagtgggagggaagaagatggctttgagcccctgcccaccaaagccgatgccctgcaggcactgtgcaccctgaggaggtggcttgaatgcaacagtgcctctccagaactctttgagaaattctatgactGTGAGGTGGAGGTGGAGCAGCTCTGCTGTCTGTGAGGAGGCATAGCTGCCTATTAGTCTCCCTCTCCTGTTTCCCATGGAAACGCCTCTCCAGAGACAGACTGGCTCTTTCCTGTGGAAATAG

This mutation is a 1343 bp deletion beginning at Chromosome 19 position 5,891,723 bp and ending after 5,893,065 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52235 CAT GGA GCT GAA CAC CAA GA Common A
52236 TGG GAC ATC TTA GAC TCA TCC A Wild type Reverse A
52237 TAA TAG GCA GCT ATG CCT CCT C Mutant Reverse A
52238 Fluorophore-1 TGG CTG AGA AGA TCC AGG TG Quencher-1 WT Probe
52239 Fluorophore-2 CCT GTG AGG TGG AGG TGG A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52235 0.40 uM
52236 0.40 uM
52237 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.