Protocol 30348: Probe Assay - Plod2<em1(IMPC)J> Probe
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

Mut= 103 bp

Wt= 105 bp

Sequence

Sequence (deletion in lower case):

 AATCAGAGGCTTTCCCATGTGATAATGAAGTGTTTAATCACcataatgttatggagaaacagcatttaatgtttggctaaaccttctttctttatcttttcagataaattattagtcataactgtcgcaacaaaagaaaatgatggattccacagatttatgaattcagccaagtatttcaattacactgtgaaggtatgatatgtctttcagtctttggacttgttggaatatgtgcctaaaatatattaactagtggtttatttaaacatgttctataagcaacagaaatgtgtatatatgtaactttcctgacttcctttggagggtcatgtccacatacattgtaatcaaggtttgacggtaactattttcactgactaaagggatggcctgctgtggtgtcagttgaaaacatgatgggacggggcTTTGGGGGTGCGCTATTGACAGTGTTTTTCCTGTTTTGTTTGGTTTGGGTTCATTGGTTGGA

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
34387 AAT CAG AGG CTT TCC CAT GT Common A
34388 Fluorophore-1 TGG AGA AAC AGC ATT TAA TGT TTG G Quencher-1 WT Probe
34389 ATC TGA AAA GAT AAA GAA AGA AGG TTT Wild type Reverse A
34390 TCC AAC CAA TGA ACC CAA AC Mutant Reverse A
34391 Fluorophore-2 TGC GCT ATT GAC AGT GTT TTT C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
34387 0.40 uM
34389 0.40 uM
34390 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.