Protocol 46348: Probe Assay - Phyhd1<em1(IMPC)J> Alt 1
Version 2.0

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

>chr2:30274680+30274767 88bp TTTCTTGGTCCCTCCTGAGA CGGCTACTCTGCTTGCTTTT

Mutant=  80 bp

Wild Type = 88 bp

Sequence

Wt Sequence: TTTCTTGGTCCCTCCTGAGAAATCTATCAACAAAATTGGCCATGgtaagcaggggcctgggagTgcagaaaagcaagcagagtagccg

Mut Sequence: ggtggcttacaaccatctgtaaatggatctgatgccctcttctgctgTGCCCCCTTgcagaaaagcaagcagagtagccg

A 592 bp deletion beginning at Chromosome 2 position 30,274,151 bp and ending after 30,274,742 bp (GRCm38/mm10). There is a 7 bp insertion (GCCCCCT)at the deletion site. 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
66280 GGT GGC TTA CAA CCA TCT GT Mutant Forward A
66281 CGG CTA CTC TGC TTG CTT TT Common A
66282 TTT CTT GGT CCC TCC TGA GA Wild type Forward A
66283 Fluorophore-1 ATT GGC CAT GGT AAG CAG G Quencher-1 WT Probe
67763 Fluorophore-2 TCT TCT GCT GTG CCC CCT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
66280 0.50 uM
66281 0.50 uM
66282 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
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.