Genotyping Protocols Database
Stock No: 004597
Protocol 25050: Standard PCR Assay - Pten<tm1Hwu>
Version 7.2

Notes

Melting curve analysis is done using a Roche Light Cycler 480.

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 = 328 bp
Heterozygote = 156 bp and 328 bp
Wild type = 156 bp

Melt Peaks
Wt = 79.7°C +/- .8
Mut = 81.7
°C +/- .8

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR9554 CAA GCA CTC TGC GAA CTG AG Forward A
oIMR9555 AAG TTT TTG AAG GCA AGA TGC Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
oIMR9554 0.50 uM
oIMR9555 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
004597 C;129S4-Ptentm1Hwu/J
006440 B6.129S4-Ptentm1Hwu/J
2 strains use this protocol