Protocol 36725: Probe Assay - Pla2g2a<em1(IMPC)J> Alternate2
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

Mutant=  70 bp

Wild Type = 82 bp

>chr4:138835157+138835238 82bp AGGCCCTCACAAGTAAAGCAAT CGCATACTTTATATTCGTCTCATTC

Sequence

Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion):

 

Mutation details

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49458 CAG CCG GGA GAT AAT AAC CA Mutant Forward A
49459 CGC ATA CTT TAT ATT CGT CTC ATT C Common A
49460 AGG CCC TCA CAA GTA AAG CAA T Wild type Forward A
49461 Fluorophore-1 CAG ACA ACT GCG TGT GTG TTC TCA Quencher-1 WT Probe
49462 Fluorophore-2 CAG CTC CGT GTC CGT TTT CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49458 0.40 uM
49459 0.40 uM
49460 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.