Genotyping Protocols Database
Protocol 32844: Probe Assay - Fgf14<Tg(tetO-MAPT*P301L)4510Kha>
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=  93 bp

Wild Type = 94 bp

This assay may be capable of distinguishing hemi from hom.  Transgene insertion site is known to be on mouse Chr 14.
This assay is designed around this insertion site, but it has not been tested on hom animals.

This assay is NOT able to be used for copy number evaluation.  If this is required, it is suggested to type by qPCR.

This protocol can be used for conventional PCR.  Omit probes and run as a separated assay.

Sequence

Wt Sequence:
gttcatatcttgaagttctcactcctattatgatgatatttggaggctgagcaTttgagaggtgaggtttatggtgaCataggagaaggattgaatgggatcacatagtgcccttaaaagaagcagtacagagagctaccctcctctgaacagtccctatacactaga
 
Mutant Sequence:
TATTCTGAATCTTAGAGTTTTTATTTGTTGAAATTTTGGACTGGATCTGTGACTTTCCAAAGTTCATATCTTGAAGTTCTCACTCCTATTATGATGATATTTGGAGGCTGAGCATTGAGAGGTGAGGTTTATGGTGatGCTGAAGGTCGCGCGCATTCCCGATGAAGAGGCCGGTTACCGCCTGTTGACATGGTG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40119 TAT TAT GAT GAT ATT TGG AGG CTG A Common A mChr14
40120 TTT TAA GGG CAC TAT GTG ATC C Wild type Reverse A mChr14
40121 Fluorophore-1 TTT ATG GTG ACA TAG GAG AAG GAT TG Quencher-1 WT Probe
40122 GGT AAC CGG CCT CTT CAT C Mutant Reverse A vector
40123 Fluorophore-2 TAT GGT GAT GCT GAA GGT CG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40119 0.40 uM
40120 0.40 uM
40122 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 4.0 -- 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.