Genotyping Protocols Database
Protocol 33897: Standard PCR Assay - Tg(APPswe,PSEN1dE9)85Dbo-Chr9
Version 1.0

Notes

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 = 142 bp
Heterozygote = 142 bp and 265 bp
Wild type = 265 bp

>chr9:112912299+112912563 265bp GTGTGATCCATTCCATCAGC GGATCTCTGAGGGGTCCAGT

 

This assay may be capable of distinguishing hemi from hom.  Transgene insertion site is known to be on mouse Chr 9.
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.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42431 GTG TGA TCC ATT CCA TCA GC Wild type Forward A
42432 GGA TCT CTG AGG GGT CCA GT Common A
42433 ATG GTA GAG TAA GCG AGA ACA CG Mutant Forward A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
42431 0.50 uM
42432 0.50 uM
42433 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
029770 B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo Apptm1Dbo/Mmjax
005864 B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax
004462 B6C3-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax
3 strains use this protocol