Stock No: 012866
Protocol 23821: Standard PCR Assay - plt (D4Mit286)
Version 1.2

Notes

FML Buffer: 500 mM KCl, 100 mM Tris HCl pH 8.3, 15 mM MgCl2, 0.01% Gelatin

Add 42 µl of loading buffer (12% ficoll 400, 0.2% bromophenol blue, 0.04 M EDTA) diluted 1:4 with TEN (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 10 mM NaCl) to PCR reaction. Load 10 µl on the gel. PCR products are separated on 3.5 % MetaPhor agarose gel with 0.5 x SYBR Green I Nucleic Acid Stain.
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

From DI:  For JR12866 use C57BL/6J as wild type control = 96 bp, plt/plt = 76 bp

Products in B6 background   Products in BALB background
plt/plt 76bp   plt/plt 76bp
wt/plt 96bp,76bp   wt/plt 76bp
wt/wt 96bp   wt/wt 76bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
10894 Fluorophore ATG GGG TCT AGG AAA ACA TGG Reverse A
10895 AAA TTA TGA GTA TTT CAC CTG AGT GTG Forward A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
10894 0.50 uM
10895 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
012866 B6N.DDD-plt/NknoJ
012873 C.DDD-plt/NknoJ
2 strains use this protocol