Protocol 33175: Probe Assay - Trank1<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

>chr9:111333275+111333369 95bp GATATGGTGTGTATGTGGAATGG TGATGATGGCATGAAGATGG

Mut= 100 bp

Wt= 95 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

TTTGACCCACACAATGGGTACAATACCCTGGAGTTCCCTGCTTATAGAGCCTGCCTCCAAGCCTGCAGAGCCCATTTCTACAGCCAGCTTCCAGGAGACTGTAATCATGGATCCTGAGAGGATATGGTGTGTATGTGGAATGGGCCATGCCACATCTGCATTTTTTGAAGccttggcaggaatgccatcccatccccatcttcatgccatcatcagctttacgttttctccagtgtaattttacatttcctctctacaaatataaaaatatttgtattgattggtgtgggatggaattaagatgtcgaaaccatttgtcaatttaaaaagtgtgagcagaatatttgggagcaagaagcaggtttgtacattgtaaagggccccgtccttttagggggttaagaggacagattttatatgcatatcagagatttcctgctaaccgctctgcctggtgacagcggtcagttccagagtcaggcccttgaggactgtgcctttgcagaatgatggctttattggtggctggtttgttcttcacaggactcctcctcgggaactggctatgctgctgtgcaacaaatccaatgcattttacaacctcgggaaatggaacgaggcttttcttgctgccaaggagtgtctccaatgggacccaacctatgtgaaggtatgggagggtggctgcttccctcttcctcctcttgatttgattgcttttttttttcctccttgtcaaagtggatattctagaaatgcttccccttgaacgcactTGCTCTTCTGAGGTCCATGGGCTGGGGGAGCCTTCCCTCCAGAGGAGCTAAGAGCAGGATAGTGAGTGTTGACACATGCTGCTGGGAGAGTGGGGCCTGACTGCCTGGTAGCTGGAGCTCAGTGAGAGGCGGGAGGAGCTCACTTAGACCTGAAGTAGTTAGACACAGATG

This mutation is a 606 bp deletion beginning at Chromosome 9 position 111,333,325 bp and ending after 111,333,930 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40821 GAT ATG GTG TGT ATG TGG AAT GG Common A
40822 TGA TGA TGG CAT GAA GAT GG Wild type Reverse A
40823 TAG CTC CTC TGG AGG GAA GG Mutant Reverse A
40824 Fluorophore-1 CTT GGC AGG AAT GCC ATC Quencher-1 WT Probe
40825 Fluorophore-2 TTG AAG TGC TCT TCT GAG GTC C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40821 0.40 uM
40822 0.40 uM
40823 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.