Protocol 36868: Probe Assay - Dpf3<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

>chr12:83315224-83315340 117bp AGTCACGCAAACGCACCA GGATCCCCTGCCAAACTAC

Mut = 111 bp

Wt = 117 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 TGAGCCAGGTGAGTAGCTGCAGAATGTCAGGACAACTTACCTAATCCTCTCTGTCTCAAGTTTTCctgctgggaaagcagggctggcttgagaacaaaggctgtgtggtgcaggtggatccactgcggccctaaagtgtgtgtgcccgtgagcctagtggccagcatagttgtaacaactgtctcgtcctcaatgtctcctcaggctcgcggctctgcaggcggaaggaggaggcatgatgccgcctctcaggaagaccacgacaaaccctacgtctgcgacagtaagtatctccttgtactgtcatagcccaggctttctgtgccactttggcagtcacgcaaacgcaccagctctggtccacgccgggctttgctgtttccagctcccggtggtcacattccctcCTGCTGTGGCGAGGCTCTGGGCATAGTAGTTTGGCAGGGGATCCCACCTCTCAGACTGCGCTCCTG

This mutation is a 342 bp deletion beginning at Chromosome 12 position 83,315,268 bp and ending after 83,315,609 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49935 GGA TCC CCT GCC AAA CTA C Common A
49936 AGT CAC GCA AAC GCA CCA Wild type Forward A
49938 GAT GAG CCA GGT GAG TAG CTG Mutant Forward A
49939 Fluorophore-1 CAC GCC GGG CTT TGC Quencher-1 WT Probe
49941 Fluorophore-2 CTC AAG TTT TCC TGC TGT GGC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49935 0.40 uM
49936 0.40 uM
49938 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.