Protocol 23419: Standard PCR Assay - Trp53<tm1Brn>
Version 2.2

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 = 390 bp
Heterozygote = 270 bp and 390 bp
Wild type = 270 bp
<p>Separated by gel electrophoresis on a 1.5% agarose gel.</p> <p><span style="color: rgb(255, 0, 0);">NE</span></p>

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR8543 GGT TAA ACC CAG CTT GAC CA Forward A
oIMR8544 GGA GGC AGA GAC AGT TGG AG Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
oIMR8543 0.50 uM
oIMR8544 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
032435 B6.129-Krastm4Tyj Trp53tm1Brn/J
008462 B6.129P2-Trp53tm1Brn/J
029971 STOCK Igs2tm1(CAG-Myc*T58A/luc)Wrey Trp53tm1Brn Rb1tm3Tyj/OlvrJ
032429 STOCK Krastm4Tyj Trp53tm1Brn Tg(Pdx1-cre/Esr1*)#Dam/J
4 strains use this protocol