Protocol 34354: Probe Assay - Togaram1<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

>chr12:64976962+64977076 115bp GAAGGCCAAGGTCACTTAAGAC GCTTTTTCACAGTATTATCCCTTTC

 

Mut= 106 bp

Wt= 115 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CCAGGACATTAGTAATCACTAATGCTTGTCTGTTAGGTTGCTCATTGCCTAATGCTGAAATGACTTAAGTAGTTTCTGCTGATGAGTCAGACATCCTTTCCTTTTGTGTTTTTTCTCCACTCTTACTGCCAgataaaagaaaggtaaagaaaagacttgaagtctagtaaaagaaggaaaaaactagcatttctatcttggaaggaaaaatactgttaatgtgtcatgctgaatgagtgtttgcattacatactcaataattctgttctttatctgggttttgtaattggttttaaattatttgttaccaaggaggcttagcaaaaatttgctaacttactctcttctcagttttcagctcatgaccttatcccgtctccaaaattaaagccctctcaaggaatgccagccagtgatgatttatgttttagcaaaaaaaggtcatcaagaaacttatttcagagtagtcgggactttaactcagagtcagttcctacatgtggtgctggtgagtaaatatttgaaataaatgctattgaagactagctaaaaagcagacactttgacataattgtgtcatgagatcataggtcatctcattgtaggctgttagtaatggaaggccaaggtcacttaagacactaatatttcttgagcataagtctacatgtcgGATATATTTCTTAATGTTCTTAATGTAGCTTTCAAGAAAGGGATAATACTGTGAAAAAGCTTGTATTCTATGTTGGAGGTGGTGGTATACACCTTTAATGCTTAGGAGGAAGGAACAAACAGATCTCTATGAGTTCCAGGCCAGGTTG

This mutation is a 542 bp deletion beginning at Chromosome 12 position 64,976,475 bp and ending after 64,977,016 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43391 GAA GGC CAA GGT CAC TTA AGA C Wild type Forward A
43392 GCT TTT TCA CAG TAT TAT CCC TTT C Common A
43393 GTC AGA CAT CCT TTC CTT TTG TG Mutant Forward A
43394 Fluorophore-1 TTC TTG AGC ATA AGT CTA CAT GTC G Quencher-1 WT Probe
43395 Fluorophore-2 CTC CAC TCT TAC TGC CAG ATA TAT TTC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
43391 0.40 uM
43392 0.40 uM
43393 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.