Protocol 35990: Probe Assay - Trappc11<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

>chr8:47527067-47527199 133bp TAACTTCTGGGAGCCCTAAGC AGAAACAAAGCACAGGTGTCAG

Mut= 122 bp

Wt= 133 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AAGCCAGAACAATTGTTCTTGCCACGTTGAGCATTCTGAGGTCTTTGGAAACTTCTTTCATGAGTGAATGCTTCCATTTTTATTTATGTTTTGGAACAGCCATGTAATATGGTTTGGGCATTATTTGTCCTGTCTATTGTGCTCAGGAGAAAATTCCACACTATTTTCTAGACAAATATGAACATATTAACTTCTGGGAGCCCTAAGCCCTCAGgaagaacaggagccctgctgccagggtgacagacttagaactttattcctgatgacaaacttgaataaatttgactacagagagctgacacctgtgctttgtttctgtttgtcaacaggggaagatgtcattgcttcggagcgggctgcggccttatgcaatgtgtgtgagctctcaggcaagtctctgtttgtgctgccccacactgaccaccttgtgggctacattataaggtaaatcagaggtttttttgttcttaagaaggaaggaaaagttggtaacttagatcctttatttacaaacaacaaagtgttcataaaaacattgtgcatggtgaagttcAGCTTGGCCAAGCACTAAATGAACAGACTGAAAGGAGTCTGAGACTAGCCTGGTCTACATTAGCAAGGTCCACTCTACCTGGGGCTACATAAGTAAGACCCTGAATCAAAAACAAACAGTGAGGATCTTCTAACACTGGCCTGATAGCCAAGCTATCTCTATAGCTACTTAGCCTGTTTTGCTTAAAAGCAGGGAAGAGTTTAATTCCAAAT

This mutation is a 342 bp deletion beginning at Chromosome 8 position 47,526,831 bp and ending after 47,527,172 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47503 TAA CTT CTG GGA GCC CTA AGC Common A
47504 AGA AAC AAA GCA CAG GTG TCA G Wild type Reverse A
47505 TAC TTA TGT AGC CCC AGG TAG Mutant Reverse A
47506 Fluorophore-1 CCT GAT GAC AAA CTT GAA TAA ATT TGA Quencher-1 WT Probe
47507 Fluorophore-2 CCT GGT CTA CAT TAG CAA GGT CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47503 0.40 uM
47504 0.40 uM
47505 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.