Protocol 47305: Probe Assay - Wee2<em3(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

Mutant=  70 bp

Wild Type = 133 bp

>chr6:40443919-40444051 133bp CTCTCAGTGTCTTCTTCACA TCCTTACCTCTGTAGATTTATAGTG

Sequence

Large deletion:  Mutant sequence with junction in uppercase

ttgagatctttgtacattcaagctggttcagttttgtatcctgtaagagttttcaaaaggctgtgttgtgtagcaaaaggccagaagtgagttcctaCAaggaggtctcggagtggaaaacactataaatctacagaggtaaggagaagaaaaaaaaaaaaaacccaacctgttgcacaacgtctgcttgccctcccctctacccaccaacagctgtgagac

This mutation is a 21,994 bp deletion of Chr6:40,443,966-40,465,959 (GRCm38/mm10)

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
70231 CTC TCA GTG TCT TCT TCA CA Wild type Forward A
70232 TCC TTA CCT CTG TAG ATT TAT AGT G Common A
70233 AAA GGC CAG AAG TGA G Mutant Forward A
70234 Fluorophore-1 ATT TCT TGT TCA GTC CCT GG Quencher-1 WT Probe
70235 Fluorophore-2 TTC CTA CAA GGA GGT CTC GG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
70231 0.40 uM
70232 0.40 uM
70233 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.