Protocol 34744: Probe Assay - Sytl5<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

>chrX:9913578+9913712 135bp GGAAGACATTCCTAACAAGGGTA ACACAGGACTAGAGTAGAGAACAAAGA

Mutant=  136 bp

Wild Type = 135 bp

Sequence

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

 

A 544 bp deletion beginning at Chromosome X position 9,913,615 bp and ending after 9,914,158 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44366 GGA AGA CAT TCC TAA CAA GGG TA Common A
44367 ACA CAG GAC TAG AGT AGA GAA CAA AGA Wild type Reverse A
44368 AGC TTC TAT GAA ATC CCA GCA Mutant Reverse A
44369 Fluorophore-1 CTG AAT ATC ACA CTA ATG AAG TCA GTA GAA A Quencher-1 MUT Probe
44370 Fluorophore-2 CCT CTC GAT TAG TTT TCC CCT Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44366 0.40 uM
44367 0.40 uM
44368 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.