Stock No: 014189
Protocol 25042: Standard PCR Assay - D11Mit8
Version 1.2

Notes

THIS STRAIN NEEDS TO BE TYPED FOR 2 MIT MARKERS.

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 5 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

For JR14189: wild = ILS and hom = ISS.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
D11Mit8-L CTT TTC ATG GAG GCA CAG GT A
D11Mit8-R TGT GAA CAG AGA CAC ACA TTC A A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
D11Mit8-L 0.50 uM
D11Mit8-R 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

This is the only strain that uses this protocol.