Protocol 28682: Standard PCR Assay - Mapt<tm1Hnd>
Version 4.2

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 = ~170 bp
Heterozygote = ~170 bp and 269 bp
Wild type = 269 bp
Separated by gel electrophoresis on a 1.5% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR7415 GCC AGA GGC CAC TTG TGT AG A
oIMR7824 AAT GGA AGA CCA TGC TGG AG A
oIMR7825 ATT CAA CCC CCT CGA ATT TT A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR7415 0.50 uM
oIMR7824 0.50 uM
oIMR7825 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
007251 B6.129X1-Mapttm1Hnd/J
017326 B6.Cg-Mapttm1Hnd Tg(MAPT)1Gds/Mmjax
015852 B6.Cg-Mapttm1Hnd Tg(MAPT*S305S)4Gds/Mmjax
017459 B6.Cg-Mapttm1Hnd Tg(MAPT*)1Gds/Mmjax
017545 B6.Cg-Mapttm1Hnd Tg(MAPT*V337M)1Gds/Mmjax
017797 B6.Cg-Mapttm1Hnd Tg(Thy1-MAPT*)3608Gds/Mmjax
017798 B6.Cg-Mapttm1Hnd Tg(Thy1-MAPT*)3610Gds/Mmjax
7 strains use this protocol