Protocol 34745: Probe Assay - Thoc7<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

>chr14:13954816-13954947 132bp TGCCAATTGTGAGGGTAGAG TTTTACGTGCATCTTGACACA

Mutant=  118 bp

Wild Type = 132 bp

Sequence

TTTTTTTCTTTTTTTGCTTCAAAAGTAGTTCTCTTAACAATTATTGACTTAGCCTTTCTGATATACCTGAAAATTTCCTCTCAACAACCCATAGAATGCCAATTGTGAGGGTAGAGTTTTACCTAAGACCTCAGCCCCTAggacaggcagatataccttggacaatcacttttctttgaagagaaaaatagaagaaagatttttaattgtgtcaagatgcacgtaaaatttatcatttttgtgttcacctcaactgatggcattaaatgtatttatttttcagtaattttcttttcattgagcaaatgagtagttatatactgttgtcttgagagttgctcagaaccaaatattcgttttaattctcattcatcttttttcctccagacgaagttatacggaaacgtcttctgattgatggagatggtgctggggatgatcggagaattaatctcctcgtgaaaagcttcattaaatggtgcaactctggatcccaagaggaagggtactttattccactgacgcttctgtatcagtatcctggtggaactttattaaggccatgttaaatagttcaatttaccttatctgtccagtgcggccacctctagactgatcagaagtttggtttatttatttgtttgtttttgttttttttttttttgttttttctgttcttctatttggaggtctcctttatgtttgtataATATGCAGATAGGACATTAGTAAAAGAAGAAATGAAGGCTTAATAATTTGATTACTTTTAGCAAAGGAAGGTTTTTTCTTTGTTAAGTTAAATTTAAGTTGCTGCAAAATAATTATATACTAATAATTACATACTGTATTAGTTAGGGTTTTACTGCTGTGCACAGACACCATGACCAAGGCAAGTCTTAT

 

A 569 bp deletion beginning at Chromosome 14 position 13,954,335 bp and ending after 13,954,903 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44371 TGC CAA TTG TGA GGG TAG AG Common A
44372 TTT TAC GTG CAT CTT GAC ACA Wild type Reverse A
44373 AAA CCT TCC TTT GCT AAA AGT AAT CA Mutant Reverse A
44374 Fluorophore-1 ACA GGC AGA TAT ACC TTG GAC AAT Quencher-1 WT Probe
44375 Fluorophore-2 AGC CCC TAA TAT GCA GAT AGG AC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44371 0.40 uM
44372 0.40 uM
44373 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.