Protocol 37038: Probe Assay - Ttk<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

>chr9:83839230+83839333 104bp ACACCCCAGAGAACTGGTTG CGCTTGACTATACCGACCAATC

Mut = 109 bp

Wt = 104 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

AGATTCACCTGCCTCTGTCCCCCCAGTGCTGGGATTAAAGGTGTGCAACACCATACCTAGCTTGAAGTTTATTCTCGCTTTTGCTATGTATGATGGAATTTCACAGCAAGAAAGCTGACACCCACAAGAAACCctgtaggttttgaaggcacggattggtgagaccaggaaattagcaaggtatttaagagaactgtagctaacttggaaaaagcacttggaaaggtaacttgtctatttctcagttgtgattttttttttctccttattcttataacttagaaactgttaaccaaattatgagggttgggaacaccccagagaactggttgaatttcttgctgaaactagagaaaaacagctcacctctaaatgacgatcttttaaataagctgattggtcggtatagtcaagcgattgaagtacttcctccagataaatacggccagaatgagagctttgctcgaatacaagtgagacttgctgaactaaaagcgtaagtattagtgaaccacacataaccaactatatatgtactatattacagagaaacagggtaaaaacaggtaatttatattttttaaactagtacttttatttttaaggtaaattttctttctgtggacatagatccctcagagttaCAGTTTGGACATTTCCAAAAATGGTACACAACTTTAATCTAGTTTTTTATTTTCACATTAAAAAAAAAAAAGAATAGTCTATCTCTTTAAAAAGAGAAGTTATCAAG

This mutation is a 511 bp deletion beginning at Chromosome 9 position 83,839,051 bp and ending after 83,839,561 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50348 ACA CCC CAG AGA ACT GGT TG Wild type Forward A
50349 CGC TTG ACT ATA CCG ACC AAT C Wild type Reverse A
50350 TGA AGT TTA TTC TCG CTT TTG C Mutant Forward A
50351 ATT AAA GTT GTG TAC CAT TTT TGG Mutant Reverse A
50352 Fluorophore-1 ACA GCT CAC CTC TAA ATG ACG ATC TTT Quencher-1 WT Probe
50353 Fluorophore-2 TGA CAC CCA CAA GAA ACC CAG TT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50348 0.40 uM
50349 0.40 uM
50350 0.40 uM
50351 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.