Genotyping Protocols Database
Protocol 23370: Standard PCR Assay - Generic Tg(APP)
Version 5.3

Notes

This assay will NOT distinguish hemizygous from homozygous transgenic animals.
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

Transgene = 377 bp
Internal positive control = 324 bp

Separated by gel electrophoresis on a 3 % agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR3610 AGG ACT GAC CAC TCG ACC AG Transgene Forward A APP
oIMR3611 CGG GGG TCT AGT TCT GCA T Transgene Reverse A APP
oIMR7338 CTA GGC CAC AGA ATT GAA AGA TCT Internal Positive Control Forward A
oIMR7339 GTA GGT GGA AAT TCT AGC ATC ATC C Internal Positive Control Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR3610 0.50 uM
oIMR3611 0.50 uM
oIMR7338 0.50 uM
oIMR7339 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles (Touchdown)
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

Stock Number Strain Name
003375 C3B6-Tg(APP695)3Dbo/Mmjax
004462 B6C3-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax
004807 B6;129-Tg(APPSwe,tauP301L)1Lfa Psen1tm1Mpm/Mmjax
005864 B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax
005866 B6.Cg-Tg(APP695)3Dbo Tg(PSEN1dE9)S9Dbo/Mmjax
006004 B6C3-Tg(tetO-APPSwInd)885Dbo/Mmjax
006005 B6.Cg-Tg(Prnp-App/APPswe)E1-2Dbo/Mmjax
006006 B6.Cg-Tg(Prnp-APP)A-2Dbo/J
007027 C57BL/6-Tg(Thy1-APPSwDutIowa)BWevn/Mmjax
007049 B6.Cg-Tg(tetO-APPSwInd)885Dbo/Mmjax
007051 B6.Cg-Tg(tetO-APPSwInd)102Dbo/Mmjax
11 strains use this protocol