Protocol 17593: Standard PCR Assay - Mapt<tm1(EGFP)Klt> Alternate 1
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 = 270 bp
Heterozygote = 270 bp and 187 bp
Wild type = 187 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
25730 CGT TGT GGC TGT TGT AGT TG Mutant Reverse A EGFP
25731 TCG TGA CCA CCC TGA CCT AC Mutant Forward A GFP fusion
oIMR3092 CTC AGC ATC CCA CCT GTA AC Wild type Forward A
oIMR3093 CCA GTT GTG TAT GTC CAC CC Wild type Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
25730 0.50 uM
25731 0.50 uM
oIMR3092 0.50 uM
oIMR3093 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
029219 B6.129S4(Cg)-Mapttm1(EGFP)Klt/J
005491 B6.Cg-Mapttm1(EGFP)Klt Tg(MAPT)8cPdav/J
004808 STOCK Mapttm1(EGFP)Klt Tg(MAPT)8cPdav/J
004779 STOCK Mapttm1(EGFP)Klt/J
4 strains use this protocol