Protocol 36311: Probe Assay - Ttc29<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

>chr8:78246026+78246110 85bp AGCCTGTCTGGTGTTTAGAGAT GAGTTCCTGTGGCTGGGAAC

Mut= 83 bp

Wt= 85 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

 AGAAATTAATGGACTTTTAATATTCAACAAGTTTTTTTTCTTGTTGGTCTTCTTACTTGGCTGGCAGGTCTGTTTGGTCTGCTGATAGGTTGGTTGGTTTGAGCAGGGGTGTGTGTAGAGGGTAGGGTAGGGAGTGGACGTGTTTGTATTTGTCTGTGTGTATGAAAAAAAAACTTTGAATAGTTAAAGTAACACATTTGTCCTTTTCAGAGGTGCGGTTACATTAACACAACTGTGTTAGGTAAGCTCTCTGCATCCTTCTAAAATGGAAGTTGCTTGATTTGTCACAAAGATCTCTTGATAGTTTTCATGCTTTCGCTGAGGAAGATGAGTAAGAAAGCCTGTCTGGTGTTTAGAGATTGAGAACCcctctctggtctaaaatggctggtgttctgctcgtgttcccagccacaggaactcatacaagaagagcatctgcgtggacatgctgcgagatgggttccacaagtccttcaccgagctctttgcactgatggaacagtgggataagctccgggaagctgcgaaggcacagtccctcttctggcagcagaggcctctggaggaccagccggacaagctagacaatttctaccactacctgaccagggccgaggctgccgagaggaaaggtaggtgctgggatgtggggttgcaccccagggaagattccaaaatgtcaccctatgaatttgatacattaagacaaatcacacagttcaacctattccactgagtggctttaaggCTAAATGACCAGCTACAAAGACATTCTGCAGATGTCATTGCAAGCCTGGGAAGACAGTGTTCTACCAAACATTTCCATAAGTTTCCAACAGGTGTAAACCTGTCCTATCATTGAGAACACATCCTGATTTGTCTTTGTGTTGTATGAATATTGACCAGTTTTCAAGGCACTGTCACATAAAAACCACACAGACATAGTTTGTTTTAGCATCTATGTAACGAGCGTTCAAAGTTTTGTCATGAGATCCTGTCAGCTTGGATTAGAAAATGGTAGTATTTAAAAAGACATGAAAAGCCTGTAATTTCTAGGGTATTGTTAAGGCTGTAATCCCAGCACTTGGGACAAAGAGACAGCTTATTCTGAGTTTGGGGTTAGCCTCAGCTACAT

This mutation is a 383 bp deletion beginning at Chromosome 8 position 78,246,056 bp and ending after 78,246,438 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48399 AGC CTG TCT GGT GTT TAG AGA T Common A
48400 GAG TTC CTG TGG CTG GGA AC Wild type Reverse A
48401 CTT CCC AGG CTT GCA ATG A Mutant Reverse A
48403 Fluorophore-1 ACC CTA AAT GAC CAG CTA CAA AGA Quencher-1 MUT Probe
48406 Fluorophore-2 CTG GTC TAA AAT GGC TGG TGT T Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48399 0.40 uM
48400 0.40 uM
48401 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.