Protocol 36795: Probe Assay - Ly6d<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

>chr15:74762488-74762603 116bp CCTTTCTGTGTGCAGTGGAG TGTGACCTCGGAACCACT

Mut = 100 bp

Wt = 116 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 CCTGATGCCCTTTTCCTTTCTGTGTGCAGTGGAGCCTCtgaatgggaacctggtgaggaaagagtgtgcaaactcatgcacctccgactacagccagcagggccatgtcagcagtggttccgaggtcacacaatgctgccagactgacctatgcaacgagaggctggtcagcgctgcacctggacatgccctgctcagcagtgtcaccctgggcctggcaacgagcctcagcctgctcacTGTTATGGCTCTCTGCCTGTAATGTAC

This mutation is a 202 bp deletion beginning at Chromosome 15 position 74,762,378 bp and ending after 74,762,579 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49735 CCT TTC TGT GTG CAG TGG AG Common A
49736 TGT GAC CTC GGA ACC ACT Wild type Reverse A
49737 GTG GGG ACA CTG ACG ACT AGA Mutant Reverse A
49738 Fluorophore-1 TGG GAA CCT GGT GAG GAA AGA G Quencher-1 WT Probe
49739 Fluorophore-2 CTC TGT TAT GGC TCT CTG CCT GTA AT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49735 0.40 uM
49736 0.40 uM
49737 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.