Protocol 36265: Probe Assay - Tbc1d21<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

>chr9:58363982-58364075 94bp GAGCCCACCCCACATTAACT CACCTTTCACCCATCTCAGC

Mut= 92 bp

Wt= 94 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AAATTCAGTCTTCAACATGAATCCTTCCAACTCAGGGGGTGCCAGAATTGTCCCCACTCTGCAGATGAGGAAACTGAGGCTTAGGGAAGGGAGCGCAACATTGCTCTGCAGGTATTCGGTTAAGTGGTGGCTCCAGAGTTCGGGGGCTACGTGGGGGGCGGGGACACTGAGCCCACCCCACATTAACTTGTCCCCACACTGTGCCAGCCAcatgttgggaacgcttgctcaaggccactcaggctgagatgggtgaaaggtggggaccaggtgggcaaggctaccccattccccttgaactcagcatcagctgctcccgaactatcccttcctggaaggggaaggtcaccttactcagccccaatgctgttctcaggaggaactataactccttgtgccagatgtatgagaaaatccagcccctgctggaaaacttacatgggaacttcacagagactcggaacaacatcggtgagtagagggagggagggagagaggaaggaagggctgggcaggggagagggagggagggctgggcaggggagagggagggagggctgggcaggggagagggagggagggctgggcaggggagagggagggagggctgggcaggggagagggagggagggaggaaggaagggctgggcaggggagagggagggagggagggctgggcaggggagagggcaggagccatttatgtcttttcctagtcatttccaggcaTAGGTGACTCCTGGGGCTTAGCAACTGCCTTGATTTTGGCCTGGGGTTTTCCTAGCCAAACCTGGGTCAATCTACCCGCCAGTGCTCATGAGTCCTACGTGTGCTTCCCTGCCACATGCTATCTCTGTTGCTTTGTCTTCTTCTCCCCACCCCTACGACTTTGAACTAAGTCAGATCTGAATTGGATGGGAGGAGCATTTGGGGGCGAGGAGAAGCCCAGAACAGCAGGGTGTGAT

This mutation is a 519 bp deletion beginning at Chromosome 9 position 58,363,515 bp and ending after 58,364,033 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48206 GAG CCC ACC CCA CAT TAA CT Common A
48207 CAC CTT TCA CCC ATC TCA GC Wild type Reverse A
48208 AAA ACC CCA GGC CAA AAT C Mutant Reverse A
48209 Fluorophore-1 AAC GCT TGC TCA AGG CC Quencher-1 WT Probe
48210 Fluorophore-2 CCA GCC ATA GGT GAC TCC TG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48206 0.40 uM
48207 0.40 uM
48208 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.