Protocol 37648: Probe Assay - Dbpht2<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

>chr12:74302199+74302291 93bp TCAGGTAGCCGAAGACATCA CAGCTGTTGAGTTAGTTGGAAAG

Mut = 88 bp

Wt = 93 bp

Fam = Mut

Hex = Wt

 

Sequence

MUT Sequence (junction in uppercase):

gattcgttccttcggaaatgctttggctggtcactgtgcctgacggttgtttgtcattagtgtccttaacCAtggttgctgtctttccaactaactcaacagctgtgcctgctgtttctt

This mutation is a 5723 bp deletion beginning at Chromosome 12 position 74,296,535 bp and ending after 74,302,257 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51852 CAG CTG TTG AGT TAG TTG GAA AG Common A
51853 TCA GGT AGC CGA AGA CAT CA Wild type Forward A
51854 ATG CTT TGG CTG GTC ACT G Mutant Forward A
51855 Fluorophore-1 AGA CAG AGC GTT CTA TAG ATA TCA GCA Quencher-1 WT Probe
51856 Fluorophore-2 TTG TTT GTC ATT AGT GTC CTT AAC CA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51852 0.40 uM
51853 0.40 uM
51854 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.