Protocol 32793: Probe Assay - Tubb4b<em1(IMPC)J>alt2
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

>chr2:25223685-25223780 96bp GTCCAACTGACTGCGGGTAG CCTGCCAATGTTCAAGGAGA

Mut= 97 bp

Wt= 96 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 

GTGCGCCTGCTCCTCCCGAAGTGCTCCTCTTCTACAGCTGTTCCGCAGTCGCCGCCGCCATGAGGGAGATCGTGCACCTGCAGGCTGGGCAGTGCGGCAACCAGATTGGCGCCAAGGTAGGAGGCTAGGTTTCTGCGAGCCAGACGCCACTGGGACAGCCAGCCGGGGAAGATGGCGGAAGcggcccggagcgtcgcccttcattgcgtcctggataccggcgtacgctgggccgagacgtagcgccggtggctggtgtcggtggggcggacggtgcgcggcccttagggattcgtcgccctggcgctcctgagactggagggagggggagggcctgccgcacccatccggggcggggctgccttggagcgcaccggccggccggcgtgactcagctaacgcccgactcccttgcagttctgggaggtaatcagcgacgagcatggcattgatcccactggcacttaccacggagatagcgacctccagctggagcgcatcaacgtgtactacaacgaagccaccggtaaggctcctctaccccatccatcctcctcaccacccttccagtttgagcacacttgacgtccccctgtcccaaccaggtggcaagtatgtgccccgcgccgtgctcgtggacttggagcccggcaccatggactcggtgcgttcagggcccttcgggcagatcttcagacctgataacttcgtctttggtgagtgtggtggaggtgtggacgggcttgctacccatgaggctcaaaggtcaaggggctgacctaaagttcttccttaatagccaccaggtgtacactttcttgccctctgagtcactcaatcccttggccgaaagcagctttaggaggaaggtccaactgactgcgggtagattagttgctagtagagagtatgagaagacctgaCCTAGTGGCAGGGGCCCTGTACTCTCCTTGAACATTGGCAGGGGTGCCTTTGGAAGTATTTACTGTCTTAGCTCTAGTGTGTTCTGTACATCTCCGTCTGGTCTCCCCCACCCCCCATCCCCAATCTATGTATCCCTGCCTGGCCTCTTTAACTGTAGACCAGGCTGACCTTAAACTCA

This mutation is a 742 bp deletion beginning at Chromosome 2 position 25,223,727 bp and ending after 25,224,468 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38908 GTC CAA CTG ACT GCG GGT AG Wild type Forward A
38909 CCT GCC AAT GTT CAA GGA GA Common A
38910 AGG TTT CTG CGA GCC AGA C Mutant Forward A
40007 Fluorophore-1 TGC TAG TAG AGA GTA TGA GAA GAC CTG Quencher-1 WT Probe
40008 Fluorophore-2 ATG GCG GAA GGC CCT AGT G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38908 0.40 uM
38909 0.40 uM
38910 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.